Cancer treatment targeting non-coding rna overexpression

ABSTRACT

Provided herein are methods directed to modulating the pro-oncogenic effects of noncoding RNAs (ncRNAs) through their interactions with specificity protein transcription factors (SpTFs). In one aspect, the disclosure provides a method of inhibiting growth of a cell, such as a transformed or cancer cell, characterized by overexpression of at least one specificity protein (Sp)-regulated ncRNA and expression of at least one Sp transcription factor (SpTF), the method comprising contacting the cell with an effective amount of an SpTF agent. In some embodiments, the ncRNA is a long noncoding RNA (lncRNA). In some embodiments, the ncRNA is a microRNA (miR). Also provided are methods of treating a cell proliferative disease, predicting the response of a subject to SpTF agent-based treatment, and monitoring the efficacy of a SpTF agent-based treatment in a subject.

CROSS REFERENCE TO RELATED APPLICATION

This application claims the benefit of U.S. Provisional Application No.61/607,984, filed Mar. 7, 2012, which is incorporated herein byreference in its entirety.

STATEMENT REGARDING SEQUENCE LISTING

The sequence listing associated with this application is provided intext format in lieu of a paper copy and is hereby incorporated byreference into the specification. The name of the text file containingthe sequence listing is 40872_SEQ_Final_(—)2013-03-07.txt. The text fileis 8 KB; was created on Mar. 7, 2013; and is being submitted via EFS-Webwith the filing of the specification.

BACKGROUND

The progression of a normal cell cycle and the transition to subsequentdevelopment of transformed cancer cells that exhibit uncontrolledgrowth, survival and metastasis/angiogenesis involves multiple geneticand epigenetic changes. For most cancers, several critical genes andpathways are responsible for the noted “characteristics” of individualtumors and cancer cells, and development of mechanism-based anticanceragents that target only one gene or pathway have had limited clinicalsuccess. In contrast, drug combinations directed to multiple targets inthe various pro-oncogenic pathways have been more efficacious.

During the past decade, the discovery of non-coding RNAs (ncRNAs) andthe characterization of their functions in both normal and cancertissues has added to the complexity of cell biology and cell signaling,providing another key element that regulates genes and is associatedwith, if not influences, cancer cell phenotype. For example, among thedifferent classes of ncRNAs, microRNAs (miRs) have been the mostextensively investigated. Functionally, miRs are small ncRNAs (21-23 bp)that exhibit sequence-specific interactions with 3′-UTR sequences intarget mRNAs and these interactions generally result in gene repressiondue to decreased translation and/or mRNA stability. It is estimatedthat >1000 miRs regulate up to 30% of all protein encoding genes.Several miRs exhibit overexpression that is associated with somedifferent tumors. Functional pro-oncogenic activity of some miRs hasbeen associated with their inhibition of multiple genes with tumorsuppressor-like activity.

Despite the advances in the art, there remains a recognized need forimproved cancer therapies, including methods of identifying optimaltreatments, and methods of monitoring the efficacy of such treatments.Specifically, there is a need to identify additional cancer targets, anddrugs that can influence them, to provide additional mechanisms toinfluence a variety targets in one or more pathways. The invention setforth in this disclosure addresses this need and provides furtheradvantages related thereto.

SUMMARY

This summary is provided to introduce a selection of concepts in asimplified form that are further described below in the DetailedDescription. This summary is not intended to identify key features ofthe claimed subject matter, nor is it intended to be used as an aid indetermining the scope of the claimed subject matter.

In one aspect, this disclosure provides a method of inhibiting growth ofa cell characterized by overexpression of at least one specificityprotein (Sp)-regulated non-coding RNA (ncRNA) and expression of at leastone Sp transcription factor (SpTF). The method comprises contacting thecell with an effective amount of an SpTF agent.

In some embodiments, the cell is a transformed cell. In someembodiments, the transformed cell is a cancer cell. In some embodiments,the cancer cell is derived from a solid tumor or non-solid tumor. Insome embodiments, the cancer cell is selected from the group consistingof a breast cancer cell, a pancreatic cancer cell, a liver cancer cell,a lung cancer cell, a prostate cancer cell, and a follicular lymphomacancer cell.

In some embodiments, the overexpression of the at least one Sp-regulatedncRNA in the cancer cell can be determined by comparing the expressionlevel in the cancer cell to a reference standard. In some embodiments,the expression level in the cancer cell to a reference standardcomprises comparing the expression level of the at least oneSp-regulated ncRNA to the expression level of the at least oneSp-regulated ncRNA in a noncancerous cell derived from the same tissue.

In some embodiments, the at least one Sp-regulated ncRNA is a longnon-coding RNA (lncRNA). In some embodiments, the lncRNA is selectedfrom the group consisting of HOTAIR, HOTAIRM, HOTTIP, MALAT-1,linc-HEIH, HULC, and AY12907. In other embodiments, the at least oneSp-regulated ncRNA is a microRNAs (miR).

In some embodiments, the at least one SpTF is Sp1, Sp3, Sp4 or otherSp/KLF transcription factor. In some embodiments, the SpTF agentcomprises: a phytochemical or derivative that induces reactive oxygenspecies (ROS) or phosphatase activity; a naturally-occurring orsynthetic triterpenoid; a non-steroidal anti-inflammatory drug (NSAID);an antisense microRNA oligonucleotide; an agent that causesoverexpression of ZBTB10, ZBTB4, or related transcriptional repressor,or that induces proteasome/caspase-dependent degradation of Sptranscription factors; a thiazolidinedione; a nitro-aspirin; anisothiocyanate; aspirin; arsenic trioxide; metformin; silibinin; or acannabinoid. In some embodiments, the Sp transcription factor agentcomprises an NSAID. In further embodiments, the NSAID is adiphenyl/diphenylamine carboxylic acid. In some embodiments, the Sptranscription factor agent comprises an phytochemical. In furtherembodiments, the phytochemical is betulinic acid or a derivative oranalog thereof. In other embodiments the phytochemical is curcumin or aderivative thereof. In some embodiments, the Sp transcription factoragent comprises a naturally-occurring triterpenoid. In furtherembodiments, the naturally-occurring triterpenoid is celastrol. In otherembodiments, the Sp transcription factor agent comprises a synthetictriterpenoid. In some embodiments, the synthetic triterpenoid is aglycyrrhetinic acid derivative. In some embodiments, the glycyrrhetinicacid derivative is methyl 2-cyano-3,12-dioxooleana-1,9-dien-28-oate ormethyl 2-cyano-3,11-dioxo-18β-olean-1,12-dien-30-oate.

In some embodiments, the method further comprises contacting the cellwith one or more small interfering RNA (siRNA) molecules that hybridizewith the mRNA encoding an SpTF under physiological or cell-cultureconditions. In some embodiments, the cell is in vivo in a subject. Inother embodiments, the cell is in vitro. In some embodiments, the cellis derived from or comprised in a sample obtained from a subject havinga cell proliferative disease or suspected of having a cell proliferativedisease, such as a cancer.

In another aspect, the disclosure provides a method of reducing theexpression of at least one specificity protein (Sp)-regulated non-codingRNA (ncRNA) in a cell that also expresses at least one Sp transcriptionfactor (SpTF), the method comprising contacting the cell with aneffective amount of an SpTF agent.

In another aspect, the disclosure provides a method of treating a cellproliferative disease, the method comprising administering to a subjectin need a therapeutically effective amount of an specificity proteintranscription factor (SpTF) agent, wherein the subject has at least onetransformed cell characterized by the overexpression of at least onespecificity protein (Sp)-regulated non-coding RNA (ncRNA) and theexpression of at least one SpTF. In some embodiments, the SpTF agent iscomprised in a pharmaceutically acceptable composition. In someembodiments, the subject is a mammal, such as selected from the groupconsisting of: human, monkey, horse, cow, sheep, goat, dog cat, mouse,rat, and guinea pig.

In another aspect, the disclosure provides a method of predicting theresponse of a subject with a cell proliferative disease to a specificityprotein transcription factor (SpTF) agent-based treatment. The methodcomprises (i) determining the expression level of at least onespecificity protein (Sp)-regulated non-coding RNA (ncRNA) in a cellsample obtained from the subject, (ii) determining the expression statusof at least one specificity protein transcription factor (SpTF) in thesame or similar cell sample obtained from the subject, and (iii)predicting a positive response of the subject with a cell proliferativedisease to treatment with an SpTF agent when the at least oneSp-regulated ncRNA is overexpressed in the cell sample and the SpTF isexpressed in the same or similar cell sample. In some embodiments, theoverexpression of the at least one Sp-regulated ncRNA is determined bycomparing the expression of the Sp-regulated ncRNA in the cell sample toa reference standard. In some embodiments, comparing the expression ofthe Sp-regulated ncRNA in the cell sample to a reference standardcomprises comparing the expression level of the at least oneSp-regulated ncRNA to the expression level of the at least oneSp-regulated ncRNA in a noncancerous cell derived from the same tissue.In some embodiments, the method further comprises administering an SpTFagent to the subject that is predicted to have a positive response totreatment.

In another aspect, the disclosure provides a method of monitoring theefficacy of a specificity protein transcription factor (SpTF)agent-based treatment in a subject with a cell proliferative disease.The method comprises: (i) determining the expression level of at leastone specificity protein (Sp)-regulated non-coding RNA (ncRNA) in a firstcell sample obtained from the subject, (ii) administering at least oneSpTF agent to the subject, and determining the expression level of theat least one Sp-regulated ncRNA in a second cell sample obtained fromthe subject. In this method, the second cell sample is obtained from thesame or similar cell source within the subject, and the second cellsample is obtained from the subject at a time after the first cellsample is obtained and after the at least one SpTF agent is administeredto the subject. The treatment is determined to be effective when theexpression level of the Sp-regulated ncRNA is lower in the second samplethan in the first sample.

In another aspect, the disclosure provides a method of evaluating acandidate specificity protein transcription factor (SpTF) agent for usein treatment of a cell proliferative disease. The method comprises:contacting a candidate SpTF agent to a cell characterized by (a)overexpression of at least one specificity protein (Sp)-regulatednon-coding RNA (ncRNA), and (b) expression of an SpTF. The methodfurther comprises determining the expression level of the at least oneSp-regulated ncRNA subsequent to the contacting step, and comparing theexpression level of the at least Sp-regulated ncRNA to a referencestandard, wherein a reduced expression level of the at leastSp-regulated ncRNA in comparison to the reference standard is indicativeof the efficacy of the candidate SpTF agent for treatment of a cellproliferative disease. In some embodiments, the cell is obtained from aplurality of similar cells, and wherein the reference standard comprisesone or more cells obtained from the same plurality of similar cells,such as a plurality of cells obtained from a tumor mass, that are notcontacted with the SpTF agent. In some embodiments, the cell is a cancercell and the reference standard comprises a noncancerous cell derivedfrom the same tissue as the cancer cell.

DESCRIPTION OF THE DRAWINGS

The foregoing aspects and many of the attendant advantages of thisinvention will become more readily appreciated as the same become betterunderstood by reference to the following detailed description, whentaken in conjunction with the accompanying drawings.

FIGS. 1A-1D illustrate HOTAIR expression and prognostic significance inpancreatic cancer. FIG. 1A illustrates HOTAIR expression in normalpancreas and pancreatic tumors from patients as determined from a datamining analysis from patient array data. FIG. 1B illustrates therelative expression of HOTAIR in pancreatic cancer patients with tumorlocalized in the pancreas (NO) or those with tumors already spread toregional lymph nodes (NO). FIG. 1C illustrates the relative expressionof HOTAIR in pancreatic cancer patients in patients with tumor detectedonly in the pancreas (T2) and tumors extending beyond the pancreas (T3).FIG. 1D illustrates HOTAIR expression in pancreatic cancer cells fromdifferent cell lines as determined by real time RT polymerase chainreaction (PCR). N indicates the number of patients.

FIGS. 2A-2D illustrate HOTAIR regulation of cancer cell growth asdetermined by RNAi knockdown of HOTAIR using two different siRNAs.Transfection of siHOTAIR oligonucleotides inhibits growth of Panc1 cells(FIG. 2A) and L3.6pL (FIG. 2B) pancreatic cells over 6 days.Transfection of siHOTAIR oligonucleotides also inhibit expression ofcyclin D1 and cyclin E, as determined by reduced mRNA levels in Panc1cells (FIG. 2C) and L3.6pL (FIG. 2D). ** indicates decrease withsignificance of p<0.05.

FIGS. 3A-3C illustrate the effects of HOTAIR on cancer cell invasion.Panc1 (FIG. 3A) and L3.6pL (FIG. 3B) cells were transfected withsiHOTAIR (I and II, separately) and after 30 hours, cell invasion wasdetermined in a Boyden chamber assay (** inhibition, p<0.05). Apoptosiswas determined by measuring enhanced Annexin V staining (FIG. 3C) (*induction, p>0.05).

FIGS. 4A-4C illustrate the effects of HOTAIR on in vivo tumordevelopment in a murine xenograft model. siHOTAIR or siCT wastransfected into L3.6pL cells, which were then used in a xenograft modelin athymic nude mice (six per group), and tumor volumes were determinedover 16 days (FIG. 4A). Tumor weights and relative HOTAIR RNA levelswere determined at the end of the study (FIG. 4B). Quantitative resultsare means±s.e. for at least three replicate determinations for each datapoint, and significant (**inhibition, p>0.05) response by siHOTAIR(compared with siCT) is indicated.

FIGS. 5A-5C illustrate the induction of gene expression for genes inPanc1 cells caused by the RNAi knockdown of HOTAIR, SP1, and EZH2. Panc1cells were transfected with siCT (control), siHOTAIR, siSP1, and siEZH2and mRNA expression of GDF15 (PRC-2 dependent) (FIG. 5A) and Mx1 andIL29 (PRC-independent) (FIGS. 5A and 5B) were determined by realtimePCR.

FIGS. 6A and 6B illustrate the knockdown effects of siRNAs for Sp1, Sp3,and Sp4 in Panc1 cells. FIG. 6A illustrates that the siSp1, siSp3, andsiSp4 oligonucleotides knockdown their respective target transcripts.FIG. 6B illustrates that each indicated siRNA also significantly reducesthe expression of lncRNAs HOTAIR, HOTTIP, and HOTARM in Panc1 cells.

FIGS. 7A and 7B illustrate the effects of CDODA-Me and CF₃CDODA on theexpression of SpTF proteins (FIG. 7A) and the HOTAIR expression (RNAlevels) in Panc1 cells (FIG. 7B). These results were obtained after 24hrs. Sp proteins were almost non-detectable after 48 hrs. * significantinhibition, p<0.05.

FIGS. 8A-8D illustrate the effects of CDODA-Me and CF₃CDODA with orwithout DDT in Panc1 cells. FIG. 8A illustrates the induction of ZBTB10expression by CDODA-Me and CF₃CDODA with or without DDT. FIG. 8Billustrates the reduction of miR27a by CDODA-Me and CF₃CDODA, whicheffect is negated by DDT. FIG. 8C illustrates the reduction of SpTFs byCDODA-Me and CF₃CDODA, which effect is negated by DDT. FIG. 8Dillustrates the reduction of HOTAIR RNA abundance by CDODA-Me andCF₃CDODA, which effect is negated by DDT.

FIGS. 9A-9D illustrate the effects of tolfenamic acid (TA) on regulationin breast cancer cells (MDA-MB-231). FIG. 9A illustrates the reductionof HOTAIR RNA expression with increasing doses of TA. FIG. 9Billustrates decreased cell proliferation with increasing dose over time.FIG. 9C illustrates downregulation of several Sp-regulated genes (EZH2,cyclin D1 and cyclin E) in MDA-MB-231 breast cancer cells withincreasing doses of TA. FIG. 9D illustrates that Sp1 knockdown by RNAiresults in decreased expression of oncogenic miRNAs that form themiR-17-92, miR-106b-25 and miR-106a-363 clusters. ** decreased withsignificance of p<0.05.

FIG. 10 illustrates the time-dependent suppression of c-Myc expressionin Panc1 cells by treatment of 6 μM CF₃DODA-Me. Reduction in c-Mycexpression was noticeable within 4 hours and c-Myc was undetectable at24 hours.

FIG. 11 illustrates the expression of lncRNA MALAT-1 in variouspancreatic cancer cell lines as determined by realtime PCR.

FIGS. 12A and 12B illustrate the effect of MALAT-1 knockdown using twosiRNAs (I and II) on Panc1 (FIG. 12A) and Panc28 (FIG. 12B) cells after48 and 72 hours. * significant inhibition, p<0.005.

FIGS. 13A and 13B illustrate the effect of MALAT-1 knockdown using twosiRNAs (I and II) on Panc28 cells. MALAT-1 knockdown induces PARPcleavage (FIG. 13A) and inhibits phosphorylation of AKT/Erk (FIG. 13B).Similar results were observed in Panc1 cells (data not shown). siMALAT-2was the most effective agent for knockdown in Panc28 cells.

FIG. 14 illustrates the reduction of MALAT-1 expression (RNA abundance)caused by knockdown of Sp1/3/4 TFs with a combined siSp1/3/4.

FIG. 15A illustrates the suppression of MALAT-1 expression (RNAabundance) caused by CDODA-Me in Panc1 and L3.6pL cells. FIG. 15Billustrates the suppression in MALAT-1 expression by CF₃DODA-Me, whicheffect is blocked by GSH. * significant decrease; ** significantreversal.

FIG. 16 illustrates the suppression of HOTTIP expression (RNA abundance)in Panc1 caused by RNAi knockdown of Sp1/3/4 TFs with a combinedsiSp1/3/4.

FIG. 17 illustrates the reduction in Panc1 and L3.6pL cell growth causedby RNAi knockdown of Sp1 and HOTTIP, as determined in a cellproliferation assay.

FIGS. 18A and 18B illustrate the regulatory effect of Sp1 and HOTTIP onvarious genes (cPARP, EZH2, Cyclin D1, surviving, and VEGF) in Panc1(FIG. 18A) and L3.6pL (FIG. 18B) cells, as determined by RNAi knockoutusing iSp1 and iHOTTIP.

FIGS. 19A and 19B illustrate the inhibition of tumor development in amurine xenograft model using L3.6pL cells cause by knockdown of HOTTIP.FIG. 19A illustrates the reduction of tumor growth caused by theknockdown as a function of time (days). FIG. 19B illustrates thereduction in tumor weight caused by the knockdown.

FIGS. 20A-20C illustrate the suppression of lncRNA expression in liverand cervical cancer cells caused by RNAi knockdown of Sp1. FIG. 20Aillustrates the significant suppression of AY1239027 and HULC expressionin HepG2 cells at 72 hours after transfection with siSp1. FIG. 20Billustrates the significant suppression of AY1239027 and HULC expressionin HeLa cells at 72 hours after transfection with siSp1. FIG. 20Cillustrates the significant suppression of AY1239027, linc-HEIH, HOTAIR,and HULC expression in HepG2 cells at 72 hours after transfection withsiSp1.

FIGS. 21A-21C illustrate the effective RNAi-based suppression of HULCexpression (i.e., reduction of RNA abundance) caused by transfectionwith siHULC for HepG2 (FIG. 21A), HeLa (FIG. 21B), and SK-(FIG. 21C)cells.

FIGS. 22A-22C illustrate the effect of HULC on cell viability for liverand cervical cancer cells as determined by RNAi knockdown of HULC usingsiHULC. siHULC was transfected in HepG2 (FIG. 22A), HeLa (FIG. 22B), andSK-Hep1 (FIG. 22C) cells and cell viability was assessed by trypan blueexclusion.

FIGS. 23A and 23B illustrate the effect of HULC knockdown on SK-Hep1cell invasion/migration, as determined by Boyden chamber cell assays.

FIGS. 24A and 24B illustrate the suppression of lncRNA expression inliver and cervical cancer cells by drug compounds that suppress Sp1.FIG. 24A illustrates the significant reduction of Sp1, AY1239027, andHULC expression 48 hours after administration of 2.5 μM CF₃DODA-Me. FIG.24B illustrates the significant reduction of Sp1, AY1239027, and HULCexpression 48 hours after administration of 1 μM CF₃DODA-Me.

FIGS. 25A-25D illustrate Sp regulation of miRs in L3.6pL and Panc28cells. L3.6pL (FIGS. 25A and 25B) and Panc28 (FIGS. 25C and 25D) cellswere transfected with iSp1, iSp3, iSp4 or iSp1/3/4 and oncogenic miR-21and miR-181b levels were determined by real time PCR.

DETAILED DESCRIPTION

Protein-encoding genes make up only a small fraction of the humangenome. Recent studies show that other regions encode for short, mediumor long non-coding RNA (ncRNAs), which are largely uncharacterized,although there has been some effort to explore their role in cancer(Cancer Res 71:3 (2011)). Recently ˜3000 human long interveningnon-coding RNAs (lncRNAs) have been identified and biologicalcharacterization studies suggest that lncRNAs have important functionsin both normal and cancer tissues. There is evidence that many lncRNAsact as molecular scaffolds that regulate molecular (protein, RNA, DNA)interactions required for various signaling networks and this isaccomplished, in part, by association with chromatin-modifyingcomplexes. HOTAIR is a 2,158 bp lncRNA localized to a boundary in theHOXC gene cluster, and HOTAIR is a negative prognostic factor for breastand liver cancer patient survival (and enhanced breast cancermetastasis) (Rinn et al., Cell (2007) 129:1311-132). RNA interferenceand overexpression studies demonstrate the pro-oncogenic activity ofHOTAIR. Increased HOTAIR expression in breast cancer patients, forexample, predicts decreased patient survival and increased metastasis,and overexpression of HOTAIR increased breast cancer cell and tumorinvasion in animal models and cell culture (Nature 464:1071 (2010)). Theactivity of HOTAIR is due, in part, to HOTAIR interactions with PolycombRepressive Complex 2 (EZH2, SUZ12 and EED) which enhances H3K27trimethylation to decrease expression of multiple genes. Other lncRNAsalso associate with PRC2 and chromatin complexes, suggesting potentialactivity similar to that described for HOTAIR.

Specificity protein 1 (Sp1) is a member of the Sp/Kruppel-like factor(KLF) family of transcription factors. These proteins are characterizedby their C-terminal domains which contain three C2H2-type zinc fingersthat recognize GC/GT boxes in promoters of target genes. The N-terminaldomains of Sp/KLF proteins are highly variable in both structure andfunction and many KLF proteins are truncated in this region. Some Sp/KLFmembers are critical for embryonic development. Knockout of Sp1, Sp3 andSp4 genes in mice results in embryo lethality or multiple developmentaldeficits. Sp and KLF proteins cooperatively interact with one anotherand other transcription factors on GC-rich promoters to activate orinhibit diverse classes of mammalian and viral genes that play acritical role in regulating cellular homeostasis.

The tissue- and age-dependent expression of Sp proteins in humans andlaboratory animal models has not been extensively investigated, however,several studies report that Sp1, Sp3 and Sp4 proteins are overexpressedin tumor vs. non-tumor tissues. For example, in gastric cancer Sp1expression was observed in tumor cells; whereas in stromal and normalglandular cells, Sp1 expression was either weak or non-detectable.Moreover, survival of gastric cancer patients increased with decreasingSp1 protein expression. Malignant transformation of human fibroblastsresulted in an 8- to 18-fold increase in Sp1 expression and thetransformed cells formed tumors in athymic nude mouse xenografts,whereas Sp1 knockdown gave cells that were non-tumorigenic in the samemouse xenograft model. Using RNA interference, it was shown that Sp1knockdown using a small inhibitory RNA (siRNA) for Sp1 (iSp1) inhibitedG0/G1 to S phase progression in MCF-7 breast cancer cells. siRNAs forSp3 (iSp3) and Sp4 (iSp4) were used along with iSp1 to show that inpancreatic cancer cells, Sp1, Sp3 and Sp4 proteins regulated expressionof vascular endothelial growth factor (VEGF), VEGF receptor 1 (VEGFR1 orFlt) and VEGFR2 (KDR) (20-22). Moreover, Sp3 acted as a repressor of p27in pancreatic cancer cells, indicating that overexpression of Spproteins in cancers contribute to their proliferative and angiogenicphenotype. However, underlying factors associated with high expressionof Sp proteins, such as Sp1, Sp3 and Sp4 in tumors are not wellunderstood.

As described in more detail below, the inventors have discovered thatSpTFs regulate many ncRNAs, and drugs that downregulate SpTFs alsodownregulate several pro-oncogenic ncRNAs. Because these ncRNAs areoften overexpressed in cancer cells, this novel discovery presentsadditional target for cancer therapies. Additionally, this discoveryprovides novel opportunities to identify cancer patients exhibitingoverexpression of ncRNAs as better candidates for therapy with SpTFdrugs than those patients exhibiting expression of Sp TF alone, topredict the response of subject to such therapy, and to monitor efficacyof treatments using such therapies.

In accordance with the foregoing, in one aspect the disclosure providesa method of inhibiting growth of a cell characterized by overexpressionof at least one specificity protein (Sp)-regulated non-coding RNA(ncRNA) and expression of at least one Sp transcription factor (SpTF).The method comprises contacting the cell with an effective amount of anSpTF agent.

In some embodiments, the cell is a transformed cell. As used herein, theterm “transformed” means a cell that comprises an alteration from ahealthy, normal state, for example, loss of an aspect of control over anormal, regulated cell-cycle. The cells can be a neoplastic cell, aprecancerous cell, or malignant neoplasm (cancer) cell. Consequent to aloss of normal cell-cycle regulation, the cell can develop and/orproliferate at an enhanced rate, thus potentially giving rise to acell-proliferative disease, such as cancer.

Cancers (or cancer cells) discussed herein can be any type of cancer,such as a carcinoma, sarcoma, lymphoma, leukemia, melanoma,mesothelioma, multiple myeloma, or seminoma. A cancer can be ametastatic cancer. The cancer cells can be derived from solid ornon-solid tumors. A cancer can be a recurrent cancer, such as recurrentliver cancer following liver transplant therapy. Cancers and cancer celltypes contemplated herein include, but are not limited to: adrenalcancer, anal cancer, bladder cancer, blood cancer, bone cancer, braincancer, breast cancer, cervical cancer, chronic or acute leukemia, CNScancer, colon cancer, cutaneous or intraocular melanoma, endocrinecancer, endometrial carcinoma, esophageal cancer, fallopian tubecarcinoma, follicular lymphoma and other non-Hodgkin's lymphomas, heador neck cancer, Hodgkin's disease, kidney cancer, larynx cancer, largeintestinal cancer, liver cancer, lung cancer, lymphocytic lymphoma,ovarian cancer, pancreatic cancer, parathyroid cancer, penile cancer,pituitary adenoma, primary CNS lymphoma, prostate cancer, rectal cancer,renal cell carcinoma, renal pelvic cancer, skin cancer, small cell lungcancer, small intestinal cancer, soft tissue tumor, spleen cancer,stomach cancer, testicular cancer, thyroid cancer, ureter cancer,urethral cancer, uterine cancer, vaginal cancer, and vulval cancer, or acombination thereof. In some embodiments, as described in more detailbelow, the cancer cells are pancreatic cancer, hepatic (liver) cancer,breast cancer, or cervical cancer.

As used herein, “inhibiting the growth” means slowing or reducing theoverall rate of cell growth and/or proliferation (i.e., division). Whilein many embodiments, the ideal scenario is to completely arrest cellproliferation, including killing the cell(s), it will be understood thatinhibition encompasses all intermediate degrees of slowing or reducingthe rates of cell growth and/or proliferation.

As used herein, “overexpress” and grammatical variants thereof refer toan expression level (of a gene) in cell or sample of cells (e.g., cell,tissue) that is greater than the level in a reference standard. One willappreciate that a reference standard can vary depending on thesituation. For example, the reference standard can include a cell orsample of cells that provide a reference expression level of the samegene. The reference standard cell(s) can be healthy cells from the samesource tissue as the target cell(s). In other embodiments, the referencestandard cell(s) can of the same type, but from a different sourcesubject(s) or determined to be healthy. In other embodiments, thereference standard can be a particular threshold established in the artthat is considered to be a normal, or typical gene expression level.Often, expression levels for any specific gene are conveyed in relativeterms in relation to control genes that are known to have relativelyconstant expression rates, such as GAPDH and Actin. Levels of expressioncan be determined according to any of many acceptable protocols known inthe art that measure the abundance of encoding RNA (e.g., mRNA), such asquantitative or semi-quantitative polymerase chain reaction (PCR),northern blot. In other embodiments applicable to protein-coding genes,the expression can be quantified in terms of amount of target proteindetected, such as by western blot. In some embodiments, overexpressionis at least twice, three, four, five, or ten times or more theexpression level of the reference standard. In some embodiments,overexpression is merely some fraction over one times the expressionlevel of the reference standard. In some embodiments, overexpressionrefers to the average expression level of several control samples.

As described above, the genome contains many regions, or genes, thatencode RNA that is not used as a template for translation to create apolypeptide. These RNA products, called “non-coding RNAs,” or “ncRNAs,”are widely variable in length and have been found to be involved in generegulation. Accordingly, in some embodiments, the ncRNA is a largenon-coding RNA (lncRNA). Nonlimiting examples of lncRNAs include HOTAIR,HOTAIRM, HOTTIP, MALAT-1, linc-HEIH, HULC, and AY12907, all of which areknown in the art. In other embodiments, the ncRNA is a microRNA (miR).Nonlimiting examples of miRs include miR-27a, miR-21, and miR-181b. See,e.g., Cell 133:217 (2008). ncRNAs encompassed by the present inventionare regulated by specificity protein transcription factors (SpTFs),which are described in more detail below. ncRNAs thus regulated can beidentified using arrays for ncRNAs and comparing their expression inuntreated cells and cells in which specificity proteins are knocked downby RNA interference (illustrative methods are demonstrated in moredetail below). Many of the arrays are commercially available (e.g.,Applied Biosystems) and can be used for screening large numbers ofncRNAs at the same time.

As indicated, HOTAIR is an exemplary a large ncRNA (lncRNA) (Cell129:1311 (2007); Nature 464:1071 (2010)). Expression determinations oflncRNAs, such as HOTAIR, can be routinely carried out by RT-PCR inpatient tissue or cells. See, e.g., FIG. 1D. (See also Yang et al., Ann.Surg. Oncol., published online February 2011; Nature 464:1071 (2010)).One can examine the relevancy of HOTAIR expression in other cancers,such as in tumors or cell lines. For example, to examine HOTAIRexpression as it relates to prognostic significance, one can performdata mining of cancer patient arrays and correlate low vs. high HOTAIRexpression and patient survival. See, additional descriptions below andNature 464:1071 (2010). To examine the functional significance of HOTAIRexpression, one can knock down HOTAIR in cells. If the cells exhibitdecreased growth and growth promoting genes and decreased invasion, thenHOTAIR functions as a tumor growth promoter and facilitates invasion.See FIGS. 2A-2D and 3A-3C herein and the accompanying descriptions. Seealso Yang et al., Ann. Surg. Oncol., published online February 2011.

As used herein, an “Sp transcription factor” or “SpTF” refers to amember of the specificity protein (Sp)/Kruppel-like factor (KLF) familyof zinc finger transcription factors. Non-limiting examples of Sptranscription factors include Sp1, Sp2, Sp3, Sp4, Sp5, Sp6, Sp7, andSp8. In some embodiments, the SpTF is one of Sp1, Sp3, and Sp4. Anystandard method of detection expression of an SpTF can by used. Suchmethods of are well-known in the art. See, e.g., U.S. Patent Appl. No.2010/0099760, incorporated herein by reference in its entirety. In someembodiments, an Sp transcription factor can be overexpressed, as definedabove.

The term “contact,” when applied to a cell, is used herein to describethe process by which an agent or composition described herein isdelivered to a target cell or is placed in direct juxtaposition with thetarget cell.

In some embodiments, a small inhibitory RNA (siRNA) or combinationthereof can be used in addition to an Sp transcription factor agentdescribed herein. Non-limiting examples of siRNAs include iSp1, iSp3,and iSp4, and combinations thereof. See, e.g., below and Mol. Cancer.Ther. 8:739 (2010).

In any embodiment herein, a cell can be in vitro, such as a cell inculture, or in vivo within a living subject. Accordingly, in someembodiments, the cell is derived from or comprised in a sample obtainedfrom a subject having a cell proliferative disease or suspected ofhaving a cell proliferative disease, such as a cancer. For example, thecell can be comprised in a sample obtained from a subject by a biopsyprocedure, and the method can be performed on it. In other embodiments,the cell can be a progeny (by cell division) of a progenitor cellobtained from a subject.

In another aspect, the disclosure provides a method of reducing theexpression of at least one specificity protein (Sp)-regulated non-codingRNA (ncRNA) in a cell that also expresses at least one Sp transcriptionfactor (SpTF), the method comprising contacting the cell with aneffective amount of an SpTF agent. As above, the cell can be in vitro,such as a cell in culture, or in vivo within a living subject.

In another aspect, the disclosure provides a method of treating a cellproliferative disease, the method comprising administering to a subjectin need a therapeutically effective amount of an specificity proteintranscription factor (SpTF) agent, wherein the subject has at least onetransformed cell characterized by the overexpression of at least onespecificity protein (Sp)-regulated non-coding RNA (ncRNA) and theexpression of at least one SpTF.

As used herein, the term “treating” includes, but is not limited to,inhibiting, slowing, or arresting the growth of cells (e.g., transformedor cancer cells), or killing the cells, or a slowing, stopping orreducing the volume or weight of a mass comprising the same. This alsoencompasses reducing the number of cells in a mass, such as a tumor.Inhibiting the growth refers to slowing or halting any increase in thesize or the number of transformed (e.g., cancer) cells or a masscomprising the same, or to halting the division of the cancer cells.Reducing the size refers to reducing the size (in terms of volume orweight) of a mass comprising the cells, or reducing the number of orsize of the same cells.

As used herein, the term “patient” or “subject” refers to a livingmammalian organism, such as a human, monkey, cow, sheep, goat, dog, cat,mouse, rat, guinea pig, or transgenic species thereof. In someembodiments, the patient or subject is a primate. Non-limiting examplesof human subjects are adults, juveniles, infants, and fetuses. A patientcan be a patient having cancer. A patient or subject can be suspected ofhaving cancer.

In some embodiments, the specificity protein transcription factor (SpTF)agents comprised in a pharmaceutically acceptable composition.“Pharmaceutically acceptable” means that which is useful in preparing apharmaceutical composition that is generally safe, non-toxic and neitherbiologically nor otherwise undesirable and includes that which isacceptable for veterinary use as well as human pharmaceutical use.

An “effective amount” of an agent or composition, generally, is definedas that amount sufficient to detectably and repeatedly achieve thestated desired result, for example, to ameliorate, reduce, minimize orlimit the extent of a disorder (e.g., a cell proliferative disease suchas cancer) or its symptoms or to increase, stimulate, or promote adesirable physiological response.

A “therapeutically effective amount” or a “pharmaceutically effectiveamount” or a “pharmacologically effective amount” means that amountwhich, when administered to a subject or patient for treating a disease,is sufficient to effect such treatment or therapy for the disease.

In another aspect, the disclosure provides a method of predicting theresponse of a subject with a cell proliferative disease to a specificityprotein transcription factor (SpTF) agent-based treatment. The methodcomprises (i) determining the expression level of at least onespecificity protein (Sp)-regulated non-coding RNA (ncRNA) in a cellsample obtained from the subject, (ii) determining the expression statusof at least one specificity protein transcription factor (SpTF) in thesame or similar cell sample obtained from the subject, and (iii)predicting a positive response of the subject with a cell proliferativedisease to treatment with an SpTF agent when the at least oneSp-regulated ncRNA is overexpressed in the cell sample and the SpTF isexpressed in the same or similar cell sample.

As used herein, the term “positive response” means that the result oftreatment includes some benefit, such the prevention, or reduction ofseverity, of symptoms, or a slowing of the progression of the disease.This can include the reduction of growth, replication and/or developmentof a transformed (e.g., cancer) cell.

In some embodiments, the overexpression of the at least one Sp-regulatedncRNA is determined by comparing the expression of the Sp-regulatedncRNA in the cell sample to a reference standard, as defined above. Insome embodiments, comparing the expression of the Sp-regulated ncRNA inthe cell sample to a reference standard comprises comparing theexpression level of the at least one Sp-regulated ncRNA to theexpression level of the at least one Sp-regulated ncRNA in anoncancerous cell derived from the same tissue. In some embodiments, themethod further comprises administering an SpTF agent to the subject thatis predicted to have a positive response to treatment.

The term “cell sample” is intended to convey that expression levels ofthe can be determined from one cell or a plurality of cells obtained inaggregate. In the latter case, the cells can have been obtained from asingle location in the subject's body, such as a biopsy sample. Thus acell sample can comprise one or more cells, or consist of a single cell.

The term “same or similar” as used in connection with cell sample isintended to mean that the cell sample can be used to determine theexpression levels of both the SpTF-regulated ncRNA and the SpTF, or theexpression levels can be determined from distinct cell samples that aresimilar. The term “similar” is intended to mean that the expressionlevel determined from one sample is understood to be meaningful to inferthe expression level of the same gene in the other sample, and viceversa. In some embodiments, the separate but similar cell samples arefrom the same tissue, and possibly from the proximate locations from thebody of the subject. In other embodiments, the cells comprising thesimilar samples are the same types of cells or are derived from the sameprogenitor cells.

The term “determining” or grammatical equivalents thereof refers toperforming a quantitative analysis.

In another aspect, the disclosure provides a method of monitoring theefficacy of a specificity protein transcription factor (SpTF)agent-based treatment in a subject with a cell proliferative disease.The method comprises: (i) determining the expression level of at leastone specificity protein (Sp)-regulated non-coding RNA (ncRNA) in a firstcell sample obtained from the subject, (ii) administering at least oneSpTF agent to the subject, and determining the expression level of theat least one Sp-regulated ncRNA in a second cell sample obtained fromthe subject. In this method, the second cell sample is obtained from thesame or similar cell source within the subject, and the second cellsample is obtained from the subject at a time after the first cellsample is obtained and after the at least one SpTF agent is administeredto the subject. The treatment is determined to be effective when theexpression level of the Sp-regulated ncRNA is lower in the second samplethan in the first sample.

An SpTF agent-based treatment is one that comprises administration of anagent that modifies, i.e., suppresses, expression or action of an SpTF.These are described in more detail below.

In this method a subject can be monitored at multiple time pointsincluding before, during initiation of treatment, and at one or moretime points after the initiation of treatment. Accordingly, at least afirst and second cell samples are obtained from the subject. In someembodiments, the at least first and second cell samples are obtainedfrom the same or similar cell source from within the body. It will beunderstood that the similarity of the cell sources (e.g., locationswithin the body) will provide cell(s) that provide a meaningfulcomparison of expression levels in a temporal context. Thus, in someembodiments, the cell(s) comprising the cell samples are of the sametype, from the same location or tissue, or are derived from the sameprogenitor cells. When a determination of reduced expression levels in asecond (or further subsequent), an inference is made that theadministration of the SpTF agents have resulted in a suppression ofncRNAs, and thus, a reduction or amelioration in their pro-oncogeniceffects.

In another aspect, the disclosure provides a method of evaluating acandidate specificity protein transcription factor (SpTF) agent for usein treatment of a cell proliferative disease. The method comprises:contacting a candidate SpTF agent to a cell characterized by (a)overexpression of at least one specificity protein (Sp)-regulatednon-coding RNA (ncRNA), and (b) expression of an SpTF. The methodfurther comprises determining the expression level of the at least oneSp-regulated ncRNA subsequent to the contacting step, and comparing theexpression level of the at least Sp-regulated ncRNA to a referencestandard, wherein a reduced expression level of the at leastSp-regulated ncRNA in comparison to the reference standard is indicativeof the efficacy of the candidate SpTF agent for treatment of a cellproliferative disease. In some embodiments, the cell is obtained from aplurality of similar cells, and wherein the reference standard comprisesone or more cells obtained from the same plurality of similar cells,such as a plurality of cells obtained from a tumor mass, that are notcontacted with the SpTF agent. In some embodiments, the cell and thereference standard comprises a noncancerous cell derived from the sametissue as the cancer cell.

Also provided are compositions comprising one or more Sp transcriptionfactor agents. For example, provided herein is a composition comprisingan amount of an Sp transcription factor agent that is effective todownregulate expression of both (i) HOTAIR, other Sp-regulated ncRNA(s),or combination thereof, and (ii) an Sp transcription factor. Suchdownregulation can result in alteration of a pro-oncogenic response inan assay system (e.g., growth, migration, invasion, and metastasis). Acomposition can comprise a therapeutically effective amount of an Sptranscription factor agent. Any composition described herein can befurther defined as a pharmaceutical composition.

The term “alkyl”, when used alone or in combination with other groups oratoms, refers to a saturated straight or branched chain consistingsolely of 1 to 6 hydrogen-substituted carbon atoms, such as 1 to 4hydrogen-substituted carbon atoms, and includes methyl, ethyl, propyl,isopropyl, n-butyl, sec-butyl, isobutyl, t-butyl, 2,2-dimethylbutyl,n-pentyl, 2-methylpentyl, 3-methylpentyl, 4-methylpentyl, n-hexyl andthe like.

The term “alkenyl” refers to a partially unsaturated straight orbranched chain consisting solely of 2 to 6 hydrogen-substituted carbonatoms that contains at least one double bond, and includes vinyl, allyl,2-methylprop-1-enyl, but-1-enyl, but-2-enyl, but-3-enyl,2-methylbut-1-enyl, 2-methylpent-1-enyl, 4-methylpent-1-enyl,4-methylpent-2-enyl, 2-methylpent-2-enyl, 4-methylpenta-1,3-dienyl,hexen-1-yl and the like.

The term “alkynyl” refers to a partially unsaturated straight orbranched chain consisting solely of 2 to 8 hydrogen-substituted carbonatoms that contains at least one triple bond, and includes ethynyl,1-propynyl, 2-propynyl, 2-methylprop-1-ynyl, 1-butynyl, 2-butynyl,3-butynyl, 1,3-butadiynyl, 3-methylbut-1-ynyl, 4-methylbut-ynyl,4-methylbut-2-ynyl, 2-methylbut-1-ynyl, 1-pentynyl, 2-pentynyl,3-pentynyl, 4-pentynyl, 1,3-pentadiynyl, 1,4-pentadiynyl,3-methylpent-1-ynyl, 4-methylpent-2-ynyl, 4-methylpent-2-ynyl, 1-hexynyland the like.

The term “aryl” refers to an aromatic mono- or bicyclic group containingfrom 6 to 14 carbon atoms that can be optionally fused with a fully orpartially saturated carbocyclic ring and can optionally be substitutedwith one or more substituents, such as one to three substituents,independently selected from C_(1-4alkyl), fluoro-substitutedC_(1-4alkyl), halo, OC_(1-4alkyl), fluoro-substituted OC_(1-4alkyl), NO₂and CN. Examples of aryl groups include phenyl, naphthyl, indanyl andthe like.

The term “heteroaryl” refers to an aromatic mono- or bicyclic groupcontaining from 5 to 14 carbon atoms, of which one to five is replacedwith a heteroatom selected from N, S, and O, that can optionally besubstituted with one or more substituents, such as one to threesubstituents, independently selected from C_(1-4alkyl),fluoro-substituted C_(1-4alkyl), halo, OC_(1-4alkyl), fluoro-substitutedOC_(1-4alkyl), NO₂ and CN. Examples of aryl groups include thienyl,benzimidazolyl, benzo[b]thienyl, furanyl, benzofuranyl, pyranyl,isobenzofuranyl, chromenyl, xanthenyl, pyrrolyl, imidazolyl, pyrazolyl,pyridyl, pyrazinyl, pyrimidinyl, pyridazinyl, indolizinyl, isoindolyl,indolyl, indazolyl, purinyl, quinolizinyl, isoquinolyl, quinolyl, andthe like.

The term “fluoro-substituted” means that, in the group being described,one or more, including all, of the hydrogen atoms has been replaced byF. For example, a fluoro-substituted alkyl includes trifluoromethyl,trifluoroethyl, pentafluoroethyl and the like.

The terms “halogen” and “halo” refer to F, Cl, Br, or I.

Under standard nomenclature rules used throughout this disclosure, thepoint of attachment of the designated side chain is described firstfollowed by the adjacent functionality toward the terminal portion. Asubstituent's point of attachment can also be indicated by a dashed lineto indicate the point(s) of attachment, followed by the adjacentfunctionality and ending with the terminal functionality.

It is intended that the definition of any substituent or variable at aparticular location in a molecule be independent of its definitionselsewhere in that molecule. It is understood that substituents andsubstitution patterns on the compounds of this invention can be selectedby one of ordinary skill in the art to provide compounds that arechemically stable and that can be readily synthesized by techniquesknown in the art as well as those methods set forth herein.

In some embodiments, the Sp transcription factor agent is selected fromthe group consisting of: (i) a phytochemical or derivative thereof thatinduces reactive oxygen species (ROS) or phosphatase activity; (ii) anaturally occurring (e.g., celastrol) or synthetic triterpenoid; (iii)an non-steroidal anti-inflammatory drug (NSAID); (iv) an antisensemicroRNA oligonucleotide; (v) an agent that causes overexpression ofZBTB10, ZBTB4, or related transcriptional repressor; (vi) athiazolidinedione; (vii) a nitro-aspirin; (viii) an isothiocyanate; (ix)aspirin; (x) arsenic trioxide; (xi) metformin; (xii) silibinin; and(xiii) a cannabinoid. In some embodiments, the Sp transcription factoragent is selected from the group consisting of: (i) an NSAID furtherdefined as a diphenyl/diphenylamine carboxylic acid; (ii) aphytochemical further defined as betulinic acid or derivative or analogthereof, or curcumin or a derivative thereof; and (iii) anaturally-occurring triterpenoid further defined as celastrol or asynthetic triterpenoid further defined as a glycyrrhetinic acidderivative. In some embodiments, the glycyrrhetinic acid derivative isfurther defined as methyl 2-cyano-3,12-dioxooleana-1,9-dien-28-oate ormethyl 2-cyano-3,11-dioxo-18β-olean-1,12-dien-30-oate.

The Sp transcription factor agent can be administered as apharmaceutically acceptable salt. The term “pharmaceutically acceptablesalt” includes both pharmaceutically acceptable acid addition salts andpharmaceutically acceptable basic addition salts. The term“pharmaceutically acceptable acid addition salt” as used herein meansany non-toxic organic or inorganic salt of any base compound of thedisclosure, or any of its intermediates. Illustrative inorganic acidswhich form suitable salts include hydrochloric, hydrobromic, sulfuricand phosphoric acids, as well as metal salts such as sodium monohydrogenorthophosphate and potassium hydrogen sulfate. Illustrative organicacids that form suitable salts include mono-, di-, and tricarboxylicacids such as glycolic, lactic, pyruvic, malonic, succinic, glutaric,fumaric, malic, tartaric, citric, ascorbic, maleic, benzoic,phenylacetic, cinnamic and salicylic acids, as well as sulfonic acidssuch as p-toluene sulfonic and methanesulfonic acids. Either the mono ordi-acid salts can be formed, and such salts can exist in either ahydrated, solvated or substantially anhydrous form. In general, the acidaddition salts of the compounds of the disclosure are more soluble inwater and various hydrophilic organic solvents, and generallydemonstrate higher melting points in comparison to their free baseforms. The selection of the appropriate salt will be known to oneskilled in the art. Other non-pharmaceutically acceptable acid additionsalts, e.g., oxalates, can be used, for example, in the isolation of thecompounds of the disclosure, for laboratory use, or for subsequentconversion to a pharmaceutically acceptable acid addition salt. The term“pharmaceutically acceptable basic salt” as used herein means anynon-toxic organic or inorganic basic addition salt of any acid compoundof the invention, or any of its intermediates, which are suitable for orcompatible with the treatment of animals, in particular humans.Illustrative inorganic bases that form suitable salts include lithium,sodium, potassium, calcium, magnesium, or barium hydroxide. Illustrativeorganic bases that form suitable salts include aliphatic, alicyclic oraromatic organic amines such as methylamine, trimethylamine and picolineor ammonia. The selection of the appropriate salt will be known to aperson skilled in the art. Other non-pharmaceutically acceptable basicaddition salts, can be used, for example, in the isolation of thecompounds of the invention, for laboratory use, or for subsequentconversion to a pharmaceutically acceptable acid addition salt. Theformation of a desired compound salt is achieved using standardtechniques. For example, the neutral compound is treated with a base ina suitable solvent and the formed salt is isolated by filtration,extraction or any other suitable method.

Sp transcription factor agents can exist in a continuum of solid statesranging from fully amorphous to fully crystalline. The term “amorphous”refers to a state in which the material lacks long range order at themolecular level and, depending upon temperature, can exhibit thephysical properties of a solid or a liquid. Typically such materials donot give distinctive X-ray diffraction patterns and, while exhibitingthe properties of a solid, are more formally described as a liquid. Uponheating, a change from solid to liquid properties occurs that ischaracterized by a change of state, typically second order (“glasstransition”). The term “crystalline” refers to a solid phase in whichthe material has a regular ordered internal structure at the molecularlevel and gives a distinctive X-ray diffraction pattern with definedpeaks. Such materials when heated sufficiently will also exhibit theproperties of a liquid, but the change from solid to liquid ischaracterized by a phase change, typically first order (“meltingpoint”).

Sp transcription factor agents can also exist in unsolvated and solvatedforms. The term “solvate” is used herein to describe a molecular complexcomprising the agent and one or more pharmaceutically acceptable solventmolecules, for example, ethanol. The term “hydrate” is employed whensaid solvent is water. A currently accepted classification system fororganic hydrates is one that defines isolated site, channel, ormetal-ion coordinated hydrates (see Polymorphism in PharmaceuticalSolids by K. R. Morris, ed. H. G. Brittain, Marcel Dekker, 1995).Isolated site hydrates are ones in which the water molecules areisolated from direct contact with each other by intervening organicmolecules. In channel hydrates, the water molecules lie in latticechannels where they are next to other water molecules. In metal-ioncoordinated hydrates, the water molecules are bonded to the metal ion.When the solvent or water is tightly bound, the complex will have awell-defined stoichiometry independent of humidity. When, however, thesolvent or water is weakly bound, as in channel solvates and hygroscopiccompounds, the water/solvent content will be dependent on humidity anddrying conditions. In such cases, non-stoichiometry will be the norm.

The present invention includes radiolabeled forms of Sp transcriptionfactor agents, for example, compounds of the invention labeled byincorporation within the structure ³H, ¹¹C or ¹⁴C or a radioactivehalogen such as ¹²⁵I and ¹⁸F. A radiolabeled compound of the inventioncan be prepared using standard methods known in the art. For example,tritium can be incorporated into a compound of the invention usingstandard techniques, for example by hydrogenation of a suitableprecursor to a compound of the invention using tritium gas and acatalyst. Alternatively, a compound of the invention containingradioactive iodo can be prepared from the corresponding trialkyltin(suitably trimethyltin) derivative using standard iodination conditions,such as [¹²⁵I] sodium iodide in the presence of chloramine-T in asuitable solvent, such as dimethylformamide. The trialkyltin compoundcan be prepared from the corresponding non-radioactive halo, suitablyiodo, compound using standard palladium-catalyzed stannylationconditions, for example hexamethylditin in the presence oftetrakis(triphenylphosphine) palladium (0) in an inert solvent, such asdioxane, and at elevated temperatures, such as 50-100° C. Further, acompound of the invention containing a radioactive fluorine can beprepared.

In some embodiments, a “derivative” refers to a chemically modifiedagent that still retains the desired effects of the agent prior to thechemical modification. As such, “derivatives,” therefore, can refer tochemically modified agents that still retain the desired effects of theparent agent prior to its chemical modification. Such effects can beenhanced (e.g., slightly more effective, twice as effective, etc.) ordiminished (e.g., slightly less effective, 2-fold less effective, etc.)relative to the parent agent, but can still be considered an Sptranscription factor agent derivative. Such derivatives can have theaddition, removal, or substitution of one or more chemical moieties onthe parent molecule. Non-limiting examples of the types of modificationsthat can be made to the compounds and structures disclosed hereininclude the addition or removal of lower unsubstituted alkyls such asmethyl, ethyl, propyl, or substituted lower alkyls such as hydroxymethylor aminomethyl groups; carboxyl groups and carbonyl groups; hydroxyls;nitro, amino, amide, imide, and azo groups; sulfate, sulfonate, sulfono,sulfhydryl, sulfenyl, sulfonyl, sulfoxido, sulfonamide, phosphate,phosphono, phosphoryl groups, and halide substituents. Additionalmodifications can include an addition or a deletion of one or more atomsof the atomic framework, for example, substitution of an ethyl by apropyl; substitution of a phenyl by a larger or smaller aromatic group.Alternatively, in a cyclic or bicyclic structure, heteroatoms such as N,S, or O can be substituted into the structure instead of a carbon atom.

Any agent or genus of agents discussed herein can be specificallyexcluded from any embodiment discussed herein.

The following is a description of Sp transcription factor (SpTF) agentsuseful in aspects of this disclosure.

As used herein, a “Sp transcription factor agent” is a molecule thatcauses downregulation or repression of an Sp transcription factor.Downregulation can occur by increasing expression of an Sp repressorgene in the cell, such as by decreasing expression of a microRNA throughcontact with the agent. The microRNA can be miR-27a. The Sp repressorgene can be ZBTB10. An agent can also induce Sp transcription factordegradation in proteasome-dependent and caspase-dependent manners. Insome embodiments, downregulation or repression results in an alterationof a pro-oncogenic response in an assay system (e.g., growth, migration,invasion, metastasis). Any agent that downregulates or represses an Sptranscription factor can be employed in embodiments herein. Sptranscription factor agents include all polymorphs and crystal formsthereof, salts, prodrugs and isomers thereof (including optical,geometric and tautomeric isomers) and isotopically-labeled agents. Insome embodiments, the molecular weight of the agent is 10,000 g/mol orless.

Numerous agents that downregulate or repress Sp transcription factorsthat are overexpressed in tumors and cancer cells have been identified,including arsenic trioxide, NSAIDs (tolfenamic acid and relatedcompounds), phytochemicals (curcumin and betulinic acid), and synthetictriterpenoids. See, e.g., Int. J. Cancer 125:1965 (2009); Cancer Res.68:5345 (2008); Carcinogenesis 30:1193 (2009); Cancer Res. 67:2816(2007); Cancer Res. 67:11001 (2007); Mol. Cancer. Res. 8:739 (2010);Exper. Cell Res. 316:2174 (2010); Mol. Pharmacol. 78:226 (2010); J.Biol. Chem. 285:25332 (2010); J. Nat'l Cancer Inst. 98:855 (2006);Cancer Res. 67:3286 (2007); Mol. Cancer. Ther. 8:533 (2009). Categoriesof Sp transcription factor agents are not necessarily mutuallyexclusive: that is, a particular agent can be a member of one or moreagent categories.

In some embodiments, the Sp transcription factor agent is anon-steroidal anti-inflammatory drug, such as diphenyl/diphenylaminecarboxylic acid (e.g., tolfenamic acid, diclofenac sodium, diflunisal),as disclosed in U.S. Publ. Appl. Nos. 2007/0259829 2008/0261911, each ofwhich is incorporated herein by reference in its entirety. In someembodiments, the agent is betulinic acid or an analog or derivativethereof as disclosed in U.S. Publ. Appl. No. 2009/0203661, incorporatedherein by reference in its entirety. The agent can act as a PPARγagonist as well as degrade Sp transcription factor(s), such as theglycyrrhetinic acid derivatives disclosed in U.S. Publ. Appl. No.2010/0099760, incorporated herein by reference in its entirety. In someembodiments, the agent is a microRNA antisense oligonucleotide, such asthose described in U.S. Publ. Appl. No. 2009/0099123, incorporatedherein by reference in its entirety. In some embodiments, the agent ismethyl 2-cyano-3,11-dioxo-18β-olean-1,12-dien-30-oate (CDODA-Me), suchas described in U.S. Publ. Appl. Nos. 2010/0099760 and 2009/0099123,noted above. In some embodiments, the agent is methyl2-cyano-3,12-dioxooleana-1,9-dien-28-oate (CDDO), as described in Mol.Pharmacol. 78:226 (2010). In some embodiments, the agent is curcumin,such as described in Cancer Res. 68:5345 (2008) and J. Biol. Chem.285:25332 (2010). In some embodiments, the agent is arsenic trioxide,such as described in Exper. Cell Res. 316:2174 (2010).

In some embodiments, the Sp transcription factor agent is a betulinicacid analog or derivative having the following formula:

wherein a bond between C1 and C2 is a single bond or a double bond; R1is H, CN, Cl, Br, F, I, CH3, CF3, OCH3, N(CH3)2, or phenyl; R2 is OH or═O; R3 is COOH, COOC_(1-4alkyl), COONH2, COONH(C_(1-4alkyl)),COON(C_(1-4alkyl))₂, or CHO; and R4 is CH(CH3)2 or C(CH3)═CH2; or apharmacologically effective salt or hydrate thereof. In someembodiments, a bond between C1 and C2 is a single bond or a double bond,R1 is H, CN, Cl, Br, F, 1, CH3, CF3, OCH3, N(CH3)₂, or phenyl; R2 is OHor ═O; R3 is COOH, COOCH3, COOCH2CH3, or CHO; and R4 is CH3, CH(CH3)2 orC(CH3)═CH2; or a pharmacologically effective salt or hydrate thereof. Inone aspect R1 can be Cl, Br or CN. In another aspect R2 can be ═O. Inyet another aspect R3 can be COOH or COOCH3. In yet another aspect C1-C2is a double bond, R1 can be CN, R2 can be ═O and R3 can be COOH orCOOCH3. In yet another aspect R1 can be Cl or Br, R2 can be ═O, and R3can be COOCH2CH3 or CHO. In some embodiments, a bond between C1 and C2is a single bond or a double bond, R1 is CN, CH3, CF3, OCH3, N(CH3)₂, orphenyl, R2 is OH or ═O, R3 is COOH, COOCH3, COOCH2CH3, or CHO, and R4 isCH(CH3)2 or C(CH3)═CH2 or comprises a pharmacologically effective saltor hydrate thereof. In some embodiments, R1 can be CN, R2 can ═O and R3can be COOH or COOCH3. Also, the substituent R3 can be COOCH2CH3 or CHOwhere R1 further comprises Cl, Br, F, or I. In some embodiments, asingle or a double bond can be formed between carbons C1 and C2. R1 canbe hydrogen, a cyano group, a halide, i.e., chlorine, bromine, fluorineor iodine, an alkyl group, e.g., methyl, a haloalkyl group, e.g.,trifluoromethyl, an alkylamine, e.g., dimethyl amine, an alkoxy group,e.g., methoxy, or a phenyl group. R2 can be hydroxy or a carbonyloxygen. R3 can be a carboxy group, an alkyl ester, e.g., a C1-C4 alkylester, preferably methyl ester or ethyl ester, an aldehyde, e.g.,formaldehyde, or an amide or alkyl substituted amide, which can befurther substituted, R4 can form the dihydro isopropyl moiety CH(CH3)2or can form the methylethylene moiety C(CH3)═CH2, as in betulinic acidor betulonic acid. R1 can be a cyano group, a chlorine or a bromine.Also, in another embodiment R2 is a carbonyl oxygen. In yet anotherembodiment, R3 is a carboxy group or the ester, particularly, the methylor ethyl ester, thereof. Alkyl substituents can be straight- orbranched-chain or cycloalkyl or, for longer chains, can be the alkenylor alkynyl derivative.

Betulinic acid analogs and derivatives can be synthesized usingwell-known and standard techniques in the chemical synthetic arts.Generally, these compounds can be, although not limited to, betulinicacid, dihydrobetulinic acid, the keto analogs betulonic acid anddihydrobetulonic acid, and derivatives thereof substituted at, althoughnot limited to, one or more of C2 or C28. In preferable non-limitingexamples, a 2-cyano group is introduced into the lupane skeleton of20(29)-dihydro betulinic acid or the corresponding methyl ester is used.

In some embodiments, an Sp transcription factor agent is aglycyrrhetinic acid derivative of the following formula:

wherein R1 is selected from CN, halo, NO2, CO2R3, C_(1-6 alkyl),fluoro-substituted C_(1-6alkyl), C_(2-6alkenyl), C_(2-6alkynyl), OR3,SR3, SOR3, SO2R3, NR3R4, C(O)NR3R4, C(O)R3, OC(O)R3, NHC(O)R3, P(O)R3R4,—C≡C—R3, —CR3═CR4R5, aryl and heteroaryl; R2 is selected fromOC_(1-6alkyl), fluoro-substituted OC_(1-6alkyl), NH2, NHC_(1-6alkyl),N(C_(1-6alkyl))(C_(1-6alkyl)), SH and SC_(1-6alkyl); R3, R4 and R5 areindependently selected from H, C₁₋₆alkyl, fluoro-substituted C₁₋₆alkyl,aryl and heteroaryl; and one of X and Y is C═O while the other is CH2,and if X is C═O then

adjacent to X represents a single bond and

adjacent to Y represents a double bond and if Y is C═O then

adjacent to Y represents a single bond and

adjacent to X represents a double bond; and pharmaceutically acceptablesalts, solvates, and prodrugs thereof. Glycyrrhetinic acid derivativescan be prepared using methods known in the art. For example, 18α- and18β-glycyrrhetinic acid and their methyl esters can be converted intothe corresponding dienones by reaction with 2-iodoxybenzoic acid as isknown (J. Amer. Chem. Soc. 124:2245 (2002)). The corresponding1-saturated-2-cyano 18β-glycyrrhetinic acid and 1-saturated-2-cyano18α-glycyrrhetinic acid and their methyl esters are known (EP 0009801)and can be reacted with 2,3-dichloro-5,6-dicyano-1,4-benzoquinone (DDQ)to give the corresponding 2-cyano-dienones. Further, dienones of 18α-and 18β-glycyrrhetinic acid and their methyl esters can be iodinated atposition 3 by reacting with iodine and pyridine in an ether solvent asdescribed in U.S. Publ. Appl. No. 2010/0099760.

In some embodiments, an Sp transcription factor agent is an antisensemicroRNA oligonucleotide. An antisense microRNA oligonucleotide can beantisense microRNA-27a. Representative examples of antisensemicroRNA-27a oligonucleotide sequences can be 5′-CCA CAC CAA GUC GUG UUCATT-3′ (set forth herein as SEQ ID NO:22) and 5′-UGA ACA CGA CUU GGU GUGGTT-3′ (set forth herein as SEQ ID NO:23). As is known in the art, anantisense RNA oligonucleotide complements all or part of the microRNAsequence and is of sufficient length that hybridization to the microRNAprevents its interaction with Sp repressor and other regulator genessuch at Myt-1 and Wee-1. Also, the antisense oligonucleotides can bestabilized using various derivatives including one or more of amorpholino group, a 2-O-methyl group and phosphorothiorate derivatives.In addition one or more of the nucleotides comprising theoligonucleotide can be modified per se. Delivery of the antisenseoligonucleotides can be achieved via cationic lipids, polymer complexes,liposomes, and other representative procedures well known and standardin the art which are effective to target and/or contact a cell ofinterest.

The following is a description of pharmaceutical formulations and routesof administration useful for aspects of this disclosure.

Pharmaceutical compositions comprise an effective amount or atherapeutically effective amount of one or more Sp transcription factoragents dissolved or dispersed in a pharmaceutically acceptable carrier.The preparation of a pharmaceutical composition that contains at leastone agent will be known to those of skill in the art in light of thepresent disclosure, as exemplified by Remington's PharmaceuticalSciences, 18th Ed. Mack Printing Company, 1990. Moreover, for animal(e.g., human) administration, it will be understood that preparationsshould meet sterility, pyrogenicity, general safety and purity standardsas required by the FDA.

As used herein, “pharmaceutically acceptable carrier” includes any andall solvents, dispersion media, coatings, surfactants, antioxidants,preservatives (e.g., antibacterial agents, antifungal agents), isotonicagents, absorption delaying agents, salts, preservatives, drugs, drugstabilizers, gels, binders, excipients, disintegration agents,lubricants, sweetening agents, flavoring agents, dyes, such likematerials and combinations thereof, as would be known to one of ordinaryskill in the art (see, for example, Remington's Pharmaceutical Sciences,18th Ed. Mack Printing Company, 1990, pp. 1289-1329). Except insofar asany conventional carrier is incompatible with the active ingredient, itsuse in the therapeutic or pharmaceutical compositions is contemplated.

Sp transcription factor agents described herein can be administered by avariety of methods, e.g., orally or by injection (e.g., subcutaneous,intravenous, intraperitoneal, etc.). Depending on the route ofadministration, an agent can be coated in a material to protect theagent from the action of acids and other natural conditions which caninactivate the agent. Agents can also be administered by continuousperfusion/infusion of a disease or wound site.

The agent can be administered parenterally, intraperitoneally,intraspinally, or intracerebrally. Dispersions can be prepared inglycerol, liquid polyethylene glycols, and mixtures thereof and in oils.Under ordinary conditions of storage and use, these preparations cancontain a preservative to prevent the growth of microorganisms.

To administer an agent by other than parenteral administration, it canbe necessary to coat the agent with, or co-administer the agent with, amaterial to prevent its inactivation. For example, the agent can beadministered in an appropriate carrier, for example, liposomes, or adiluent. Pharmaceutically acceptable diluents include saline and aqueousbuffer solutions. Liposomes include water-in-oil-in-water CGF emulsionsas well as conventional liposomes.

Pharmaceutical compositions suitable for injectable use include sterileaqueous solutions (where water soluble) or dispersions and sterilepowders for the extemporaneous preparation of sterile injectablesolutions or dispersion. In all cases, the composition must be sterileand must be fluid to the extent that easy syringability exists. It mustbe stable under the conditions of manufacture and storage and must bepreserved against the contaminating action of microorganisms such asbacteria and fungi. The carrier can be a solvent or dispersion mediumcontaining, for example, water, ethanol, polyol (such as, glycerol,propylene glycol, and liquid polyethylene glycol, and the like),suitable mixtures thereof, and vegetable oils. The proper fluidity canbe maintained, for example, by the use of a coating such as lecithin, bythe maintenance of the required particle size in the case of dispersionand by the use of surfactants. Prevention of the action ofmicroorganisms can be achieved by various antibacterial and antifungalagents, for example, parabens, chlorobutanol, phenol, ascorbic acid,thimerosal, and the like. In many cases, it will be preferable toinclude isotonic agents, for example, sugars, sodium chloride, orpolyalcohols such as mannitol and sorbitol, in the composition.Prolonged absorption of the injectable compositions can be brought aboutby including in the composition an agent which delays absorption, forexample, aluminum monostearate or gelatin.

Sterile injectable solutions can be prepared by incorporating the Sptranscription factor agent in the required amount in an appropriatesolvent with one or a combination of ingredients enumerated above, asrequired, followed by filtered sterilization. Generally, dispersions areprepared by incorporating the agent into a sterile carrier whichcontains a basic dispersion medium and the required other ingredientsfrom those enumerated above. In the case of sterile powders for thepreparation of sterile injectable solutions, the preferred methods ofpreparation are vacuum drying and freeze-drying which yields a powder,plus any additional desired ingredient from a previouslysterile-filtered solution.

An agent can be orally administered, for example, with an inert diluentor an assimilable edible carrier. The agent and other ingredients canalso be enclosed in a hard or soft shell gelatin capsule, compressedinto tablets, or incorporated directly into a subject's diet. For oraltherapeutic administration, the agent can be incorporated withexcipients and used in the form of ingestible tablets, buccal tablets,troches, capsules, elixirs, suspensions, syrups, wafers, and the like.The percentage of the agent in the compositions and preparations may, ofcourse, be varied. The amount of the agent in therapeutically usefulcompositions (e.g., pharmaceutical compositions) is such that a suitabledosage will be obtained.

It is especially advantageous to formulate parenteral compositions indosage unit form for ease of administration and uniformity of dosage.“Dosage unit form” as used herein refers to physically discrete unitssuited as unitary dosages for the subjects to be treated, each unitcontaining a predetermined quantity of agent calculated to produce thedesired therapeutic effect in association with the requiredpharmaceutical carrier. The specification for the dosage unit forms ofthe invention are dictated by and directly dependent on (a) the uniquecharacteristics of the agent and the particular therapeutic effect to beachieved, and (b) the limitations inherent in the art of compoundingsuch an agent for the treatment of a selected condition in a patient.

The agent can also be administered topically to the skin, eye, ormucosa. Alternatively, if local delivery to the lungs is desired theagent can be administered by inhalation in a dry-powder or aerosolformulation.

Agents are typically administered at a dosage sufficient to treat acondition associated with a condition in a patient. The actual dosageamount of an agent or composition comprising an agent administered to asubject can be determined by physical and physiological factors such asage, sex, body weight, severity of condition, the type of disease beingtreated, previous or concurrent therapeutic interventions, idiopathy ofthe subject and on the route of administration. These factors can bedetermined by a skilled artisan. The practitioner responsible foradministration will typically determine the concentration of agent(s) ina composition and appropriate dose(s) for the individual subject. Thedosage can be adjusted by the individual physician in the event of anycomplication.

An effective amount or therapeutically effective amount can vary fromabout 0.001 mg/kg to about 1,000 mg/kg in one or more doseadministrations daily, for one or several days (depending, of course, ofthe mode of administration and the factors discussed above). In someembodiments, the amount is less than 10,000 mg per day. In anothernon-limiting example, a dose can comprise from about 1 microgram/kg/bodyweight of agent to about 1,000 mg/kg/body weight or more peradministration, and any range derivable therein. In some embodiments, apharmaceutical composition can comprise, for example, at least about0.1% of an Sp transcription factor agent. In other embodiments, an agentcan comprise between about 2% to about 75% of the weight of thepharmaceutical composition unit.

Single or multiple doses of agents are contemplated. Desired timeintervals for delivery of multiple doses can be determined by one ofordinary skill in the art employing no more than routineexperimentation. As an example, subjects can be administered two dosesdaily at approximately 12 hour intervals. In some embodiments, the agentis administered once a day. The agent(s) can be administered on aroutine schedule. As used herein a routine schedule refers to apredetermined designated period of time. The routine schedule canencompass periods of time which are identical or which differ in length,as long as the schedule is predetermined. For instance, the routineschedule can involve administration twice a day, every day, every two,three, four, five, or six days, or on a weekly or monthly basis, or anyset number of days or weeks there-between. Alternatively, thepredetermined routine schedule can involve administration on a twicedaily basis for the first week, followed by a daily basis for severalmonths, etc. In other embodiments, the invention provides that theagent(s) can taken orally and that the timing of which is or is notdependent upon food intake. Thus, for example, the agent can be takenevery morning and/or every evening, regardless of when the subject haseaten or will eat.

In addition to being used as a monotherapy, the agents described hereincan also find use in combination therapies. Effective combinationtherapy can be achieved with a single composition that includes both anSp transcription factor agent and a second therapeutic agent, or withtwo distinct compositions, at the same time, wherein one compositionincludes the Sp transcription factor agent according, and the otherincludes the second therapeutic agent(s). Alternatively, the therapy canprecede or follow the other agent treatment by intervals ranging fromminutes to months. Administration of the compounds of the presentinvention to a patient will follow general protocols for theadministration of pharmaceuticals, taking into account the toxicity, ifany, of either agent. It is expected that the treatment cycles would berepeated as necessary. For the treatment or prevention of cancer, Sptranscription factor agents can be combined with one or more of thefollowing: radiation, chemotherapy agents, or vaccine therapies designedto promote an enhanced immune response targeting cancer cells. Further,drugs that downregulate Sp transcription factors decrease “resistance”genes such as survivin and enhance drug- and drug-radiation therapies.See, e.g., Mol. Cancer. Ther. 8:533 (2009).

It will be understood that any embodiment, characteristic, element,definition, or general description provided for any aspect of thedisclosure can be applied to any other aspect of the disclosure withoutlimitation, unless explicitly stated. Thus, any embodiment discussedherein can be implemented with respect to any method, agent, orcomposition of the invention, and vice versa. Furthermore, agents andcompositions of the invention can be used to achieve methods of theinvention.

The use of the word “a” or “an,” when used in conjunction with the term“comprising” herein can mean “one,” but it is also consistent with themeaning of “one or more,” “at least one,” and “one or more than one.”

Throughout this application, the term “about” is used to indicate that avalue includes the inherent variation of error for the device, themethod being employed to determine the value, or the variation thatexists among the study subjects.

The terms “comprise,” “have” and “include” are open-ended linking verbs.Any forms or tenses of one or more of these verbs, such as “comprises,”“comprising,” “has,” “having,” “includes” and “including,” are alsoopen-ended. For example, any method that “comprises,” “has” or“includes” one or more steps is not limited to possessing only those oneor more steps and also covers other unlisted steps.

As an alternative to or in addition to “comprising,” any embodimentherein can recite “consisting of:” The transitional phrase “consistingof” excludes any element, step, or ingredient not specified in theclaim.

The following is a description of the establishment of HOTAIR as anegative prognostic factor in pancreatic cancer and an analysis ofHOTAIR pro-oncogenic activities. This description is based on Kim, K.,et al., “HOTAIR Is a Negative Prognostic Factor and ExhibitsPro-Oncogenic Activity in Pancreatic Cancer,” Oncogene (epub can 2012),which is incorporated herein by reference in its entirety.

Introduction:

Studies on the human, mouse and other genomes have demonstrated that alarge number of genes and their corresponding RNAs are non-coding RNAs(ncRNAs) that are differentially expressed in various organs andtissues. The functions of some ncRNAs have been characterized and it isevident that they are the key factors in gene regulation and influencenormal and cancer cell phenotypes. Among the different classes ofncRNAs, microRNAs have been the most extensively investigated, and it isestimated that 41000 microRNAs regulate up to 30% of allprotein-encoding genes. Several microRNAs are overexpressed in differenttumors and their functional pro-oncogenic activity is usually associatedwith induction of oncogenes or inhibition of multiple genes with tumorsuppressor-like activity.

Rinn and coworkers have identified up to 3000 human long interveningnon-coding RNAs (lncRNAs), (Rinn, J. L., et al., “Functional Demarcationof Active and Silent Chromatin Domains in Human HOX Loci by NoncodingRNAs,” Cell 129:1311-1323, 2007; Khalil, A. M., et al., “Many HumanLarge Intergenic Noncoding RNAs Associate With Chromatin-ModifyingComplexes and Affect Gene Expression,” Proc. Nat'l Acad. Sci. USA106:11667-11672, 2009) and biological characterization studies suggestthat lncRNAs have important functions in both normal and cancer tissues.There is evidence that many lncRNAs act as scaffolds that regulatemolecular (protein, RNA and DNA) interactions required for varioussignaling networks and this is accomplished, in part, by associationwith chromatin-modifying complexes. HOTAIR is a 2158-bp lncRNA localizedto a boundary in the HOXC gene cluster. HOTAIR is a negative prognosticfactor for breast, colon and liver cancer patient survival, andincreased HOTAIR expression in patients has been correlated withenhanced breast and colon cancer metastasis. HOTAIR expression has beenlinked to increased breast, liver and colon cancer cell invasiveness,whereas RNA interference (RNAi) studies in liver cancer cells showedthat HOTAIR alone had minimal effects alone on cell viability orapoptosis but enhanced the activities of other agents. The activity ofHOTAIR is due, in part, to interaction of HOTAIR with the PolycombRepressive Complex 2 (PRC2) (EZH2, SUZ12 and EED), which enhances H3K27trimethylation to decrease expression of multiple genes. Other lncRNAsalso associate with PRC2 and other chromatin complexes, suggestingpotential gene-repressive activity similar to that described for HOTAIR.

In this study, data-mining studies of publicly available databasesshowed that HOTAIR was overexpressed in pancreatic tumors compared withthe normal pancreas and was more highly expressed in advanced tumors.HOTAIR exhibited variable expression in pancreatic cancer cells, andknockdown of HOTAIR in Panc1 and L3.6pL pancreatic cancer cells by RNAishowed that HOTAIR was associated with enhanced cell invasion, cellproliferation, modulation of cell cycle progression and induction ofapoptosis. HOTAIR knockdown in L3.6pL cells also inhibited tumor growthin a mouse xenograft model. Knockdown of HOTAIR in Panc1 cells resultedin significant (>1.5) changes in expression of 1006 genes and analysisof the data suggested that HOTAIR mediated gene regulation has acritical role in pancreatic cancer progression and will be a novelepigenetic molecular target for treating pancreatic cancer patients.

Methods and Materials:

Cell lines: Human pancreatic cancer cell lines Panc1, MiaPaCa2 andPanc28 were obtained from American Type Culture Collection (Manassas,Va.). L3.6 pl pancreatic cancer cell line was kindly provided from Dr.I. J. Fidler in M.D. Anderson Cancer Center (Houston, Tex.). The cancercell lines were grown and maintained in Dulbecco's modified Eagle'smedium (DMEM) nutrient mixture (Hyclone, Logan, Utah) supplemented with0.22% sodium bicarbonate, 0.011% sodium pyruvate, 10% fetal bovine serum(FBS), and 10 ml/l 100× antibiotic antimycotic solution (Sigma Aldrich,St. Louis, Mo.).

Gene set enrichment analysis (GSEA): Pancreatic cancer patient geneprofiling data (GSE20501) was obtained from Gene Expression Omnibus(GEO) site. The patients are classified into two groups according totheir HOTAIR expression level (top 15%: high vs. bottom 85%: low) andGSEA was carried out to assess the effects of HOTAIR expression level onvarious biological pathways using these two classified data sets.Similarly, GSEA was also performed using gene profiling data setsobtained from control siRNA control vs. HOTAIR siRNA (siHOTAIR I)transfected Panc1 cells. Significantly enriched biological pathways wereidentified, which produced nominal p-value <0.05 and false discoveryrates (FDR)<0.25.

RNA isolation and quantitative PCR: Total RNA was extracted either frompancreatic cancer cell lines or from tissue samples. Five control andsiRNA HOTAIR-transfected tissue samples were analyzed, respectively,from xenograft study using mirVana RNA isolation kit from Ambion(Austin, Tex., USA), and quantitative PCR was carried out using iCyclerIQTM real-time PCR detection system (Bio-Rad, Hercules, Calif., USA)after reverse transcription to cDNAs. The RT primer sets for HOTAIR,PCDHB5, PCDH10, JAM2, LAMB3, ABL2, SNAI and LAMC2 were used as describedpreviously (Gupta, R. A., et al., “Long Non-Coding RNA HOTAIR ReprogramsChromatin State to Promote Cancer Metastasis,” Nature 464:1071-1076,2010). The forward and reverse oligonucleotide primer sequencesspecifically for HOTAIR amplification and detection are also set forthherein as SEQ ID NOS:1 and 2, respectively. All other primer sets andsequences for this assay are shown in TABLE 1.

TABLE 1 Primer set sequences for quantitative RT-PCR analysis Primer SetForward Reverse GDF15 GGGCAAGAACTCAGGACGG TCTGGAGTCTTCGGAGTGCAA(SEQ ID NO: 24) (SEQ ID NO: 25) IL-29 TATGTGGCCGATGGGAACCTAGGGTGGGTTGACGTTCTCA (SEQ ID NO: 26) (SEQ ID NO: 27) OAS-1TGTCCAAGGTGGTAAAGGGTG CCGGCGATTTAACTGATCCTG (SEQ ID NO: 28)(SEQ ID NO: 29) MX1 GTTTCCGAAGTGGACATCGCA CCATTCAGTAATAGAGGGTGGGA(SEQ ID NO: 30) (SEQ) ID NO: 31) IFTM1 TCGCCTACTCCGTGAAGTCTATGAGGATGCCCAGAATCAG (SEQ ID NO: 32) (SEQ ID NO: 33) IL28ACACCCTGCACCATATCCTCT CACTGGCAACACAATTCAGG (SEQ ID NO: 34)(SEQ ID NO: 35) IL28B CTGCTGAAGGACTGCAAGTG GAGGATATGGTGCAGGGTGT(SEQ ID NO: 36) (SEQ ID NO: 37)

siRNA transfection and luciferase assay: Pancreatic cancer cells weretransfected with 100 nM of control siRNA or HOTAIR siRNA I (availablefrom Sigma Aldrich, catalogue No. SASI_Hs02_(—)00380445) or HOTAIR siRNAII (available from Sigma Aldrich, catalogue No. SASI_Hs02_(—)00380446)using Lipofectamine 2000 (Invitrogen, Grand Island, N.Y., USA). Panc1cells were cotransfected with control siRNA or HOTAIR siRNA I and HOTAIRII, and various length of GDF15 luciferase constructs. Luciferaseactivities were measured after 24 h as described previously. MISSIONsiRNAs for EZH2 and Suz12 were also purchased from Sigma Aldrich.

Chromatin immunoprecipitation assay: Antibodies for RNA polymerase, EZH2and Histone H3 trimethyl Lys 27 antibodies were obtained from ActiveMotif (Carlsbad, Calif., USA), and ChIP assay was performed as describedpreviously. The ChIP primer sequences (forward)5′-GGAGCACCCTGCTTAGACTG-3′ (set forth herein as SEQ ID NO:38) and(reverse) 5′-GGGCCTCAGTATCCTCTTCC-3′ (set forth herein as SEQ ID NO:39)were used to amplify the 5′ promoter region (−680 to −190) of the GDF 15gene.

Boyden chamber cell invasion and apoptosis assays: Pancreatic cancercells were transfected with either siRNAs (HOTAIR vs. control) orexpression vectors (empty vs. HOTAIR) and cell invasion assay wasperformed as described previously. Cells were transfected with siCT orsiHOTAIR and after 72 h, cells were stained for Annexin V using theVibrant apoptosis assay kit as described.

Cell proliferation and fluorescence-activated cell sorting analysis andapoptosis: Cells were seeded in 12-well plates and transfected witheither appropriate siRNA or expression vectors, and cell numbers werecounted at the indicated times using a Coulter Z1 cell counter (BeckmanCoulter, Fullerton, Calif., USA). For fluorescence-activated cellsorting analysis, after pancreatic cancer cells were transfected withsiRNAs for either control or HOTAIR, cells were stained with propidiumiodide solution and were analyzed on a FACS Calibur Flow Cytometer(Becton Dickinsin Systems, Franklin Lakes, N.J., USA).

Xenograft study: L3.6pL cells (6×10⁴ ml) were transfected with 100 nMsiHOTAIR or siCT using Lipofectamine. After 48 h cells were collectedand 1×10⁶ cells injected into either side of the flank area of femalenude mice (Harlan), and tumor volumes and weights were determined inmice from the siHOTAIR (six mice) or siCT (six mice) groups asdescribed, and siHOTAIR levels were determined by realtime PCR. Tumorvolumes were measured (0.5×length×width²) and after 16 days, the micewere killed and tumor weights were measured and also used for furtheranalysis as described.

Immunohistochemistry and TUNEL assay: Tissue sections weredeparaffinized in xylene and treated with graded series of alcohol andrehydrated in PBS. Antigen retrieval was done using 10 mM sodium citrate(pH 6.0-6.2) and endogenous peroxidase was blocked by 3% hydrogenperoxide in methanol for 6 min. Slides were then incubated with blockingserum (Vecstatin ABC Elite kit, Vector Laboratories, Burlingame, Calif.,USA) for 45 min. Samples were then incubated overnight with Ki-67 andPCNA antibodies at 4° C. Sections were then washed in PBST and thenincubated with biotinylated secondary antibody followed by streptavidin.The brown staining specific for antibody binding was developed byexposing the avidin and biotinylated peroxidase complex todiaminobenzidine reagent (Vector Laboratories) and sections were thencounterstained with hematoxylin (Vector Laboratories). The in situ celldeath detection POD kit was used for the TUNEL assay according to theinstructions in the protocol for tissue sections.

Statistical analysis: Statistical significance of differences betweendifferent groups was determined using Student's t-test. Gene profilingdata were analyzed either by BRB-Array Tools44 or by Gene Patternsoftware. Cluster and Treeview programs were employed for generation ofheat maps and for gene clustering. Kaplan-Meier analysis and log-ranktest were applied to evaluate the prognostic significance of HOTAIRexpression level in terms of patient survival. Cox proportional hazardregression model was also used to evaluate independent prognosticfactors correlated with tumor stage and lymph node metastasis.

Results:

New data mining analyses of gene profiling databases (Stratford, J. K.,et al., “A Six-Gene Signature Predicts Survival of Patients WithLocalized Pancreatic Ductal Adenocarcinoma,” PLoS Med. 7:e1000307, 2010;Badea, L., et al., “Combined Gene Expression Analysis of Whole-Tissueand Microdissected Pancreatic Ductal Adenocarcinoma Identifies GenesSpecifically Overexpressed in Tumor Epithelia,” Hepatogastroenterology55:2016-27, 2008; Collisson, E. A., et al., “Subtypes of PancreaticDuctal Adenocarcinoma and Their Differing Responses to Therapy,” Nat.Med 17:500-503, 2011) showed that among 36 pancreatic cancer patients,HOTAIR was more highly expressed in pancreatic tumors compared tonon-tumor tissue (FIG. 1A). Additionally, HOTAIR was more highlyexpressed in tumors spread to regional lymph nodes (N1) compared totumors localized only in the pancreas (NO) (FIG. 1B). HOTAIR was alsomore highly expressed in tumors extending beyond the pancreas (T3)compared to tumors only detected in the pancreas (T2) (FIG. 1C).Kaplan-Meier survival analyses also demonstrated that patients with lowHOTAIR expression (bottom 85%) had significantly increased overallsurvival compared to patients with high HOTAIR expression (top 15%).Furthermore, the results of Cox proportional hazard regression analysesconsistently indicated that HOTAIR levels and N stage are stronglycorrelated with overall patient survival (p<0.05). Gene set enrichmentanalyses (GSEA) using pancreatic patient gene profiling data (GSE21501)demonstrated that gene set differences in HOTAIR high vs. low patientsindicated that HOTAIR regulates gene sets mainly associated with cellproliferation and cell cycle progression (not shown). These resultsdemonstrate that HOTAIR is involved in mechanisms underlying theprogression of the disease and is a negative prognostic factor forpancreatic cancer. FIG. 1C illustrates relative HOTAIR RNA levels invarious pancreatic cancer cell lines, adjusted for GAPDH. Theseexpression data demonstrate that HOTAIR was highly expressed in Panc1and L3.6pL cells, whereas lower expression was observed in Panc-28,MiaPaca-2 and AsPC-1, and BxPC3 cells. These results were consistentwith data mining from array data from several pancreatic cancer celllines, which also showed that HOTAIR was overexpressed in Panc1 cells(not shown).

Panc1 cells are a highly aggressive ‘basal-like’ pancreatic cancer cellline, and knockdown of HOTAIR by RNAi (siHOTAIR) resulted in changes inexpression (41.5-fold) of 1006 genes, in which expression of 454 geneswas enhanced and expression of 552 genes was decreased (not shown).HOTAIR knockdown was performed using two different siRNAs (siHOTAIR Iand siHOTAIR II, see SEQ ID NOS:22 and 23, respectively) to avoidoff-target effects, each exhibiting effective and specific knockdown ofHOTAIR. Results from GSEA using gene ontology terms showed that 20significant gene sets were affected by HOTAIR knockdown in Panc1 cellscompared with control (siCT) cells. As 10 out of the 20 gene ontologyterms were related to the cell cycle, it was concluded that HOTAIRregulation of cell viability and cell cycle progression was important inboth cultivated pancreatic cancer cells and patients.

Previous studies characterized HOTAIR-regulated genes by overexpressionof this ncRNA in MDA-MB-231 breast cancer cells. There were 241 commongenes among 1006 and 9260 genes (>1.5-fold change) modulated by HOTAIRknockdown or overexpression in Panc1 and MDA-MB-231 cancer cells,respectively. As the HOTAIR-regulated genes were determined by oppositeprocedures (knockdown vs. overexpression) in Panc1 and MDA-MB-231 cells,respectively, heat maps of the genes induced or repressed by HOTAIRknockdown or overexpression in both cell lines were compared. The heatmap illustrates the limited overlap between the 241 genes regulated byHOTAIR in Panc1 and MDA-MB-231 cells, and this is further evidenced thatamong the 119 genes induced in Panc1 cells by siHOTAIR II, only 27 geneswere repressed in MDA-MB-231 cells by HOTAIR overexpression, whereasamong the 122 genes repressed after HOTAIR KO in Panc1 cells only 24genes were induced (not shown). Moreover, siHOTAIR resulted in therepression of 122 genes in Panc1 cells, and only 18 of these genes wereinduced by HOTAIR overexpression in MDA-MB-231 cells. Thus, HOTAIR hasan important role in PRC2-mediated gene suppression. Comparison ofHOTAIR-regulated genes in Panc1 cells with HOTAIR/PRC2-coregulated genesin MDA-MB-231 (in a chromatin immunoprecipitation assay (ChIP) assay),indicated only nine genes in common but only minimal overlap betweenPRC2-regulated genes induced by siHOTAIR in Panc1 cells and genessuppressed by HOTAIR overexpression in MDA-MB-231 cells. The differencesin HOTAIR-regulated genes in Panc1 and MDA-MB-231 cells were furtherinvestigated by comparing specific genes repressed and induced by HOTAIRoverexpression in the latter cell line. Among three genes repressed byHOTAIR overexpression in MDA-MB-231 cells (PCDHB5, PCDH10 and JAM2), twowere enhanced after HOTAIR knockdown in Panc1 and L3.6pL cells, but onlyJAM2 expression was enhanced in both cell lines (not shown). LAMB3,ABL2, SNA1 and LAMC2 were induced by HOTAIR overexpression in MDA-MB-231cells; however, none of these genes were repressed by siHOTAIR in eitherPanc1 or L3.6pL cells (FIG. 2D), and two of the four genes (ABL2 andSNAI) were induced in both cell lines. These results confirm that HOTAIRregulates significantly different sets of genes in pancreatic vs. breastcancer cells.

HOTAIR knockdown induces or represses multiple genes that couldcontribute to the functional pro-oncogenic activity of HOTAIR in Panc1cells. Expression of seven genes with tumor suppressor-like activitythat are constitutively suppressed by HOTAIR and induced in Panc1 cellswere examined after transfection with siHOTAIR. siHOTAIR I significantlyinduced expression of GDF15, IL29, IL28A, IL28B, IFTM1, OAS1, and MX1mRNA in Panc1 and L3.6pL cells. The role of the PRC2 complex incoregulating suppression of these HOTAIR-suppressed genes wasinvestigated by EZH2 knockdown (siEZH2), and only GDF15 mRNA expressionwas induced in Panc1 and L3.6pL cells transfected with siEZH2. Similarresults were observed for knockdown of Suz12, another member of the PRC2complex. GDF15 is a growth-inhibitory/proapoptotic gene, and ChIPanalysis demonstrates that primers directed at the proximal region ofthe GDF15 promoter detected H3K27 trimethylation and EZH2 binding butnot RNA pol II interactions in Panc1 cells transfected with siCT(control). In contrast, knockdown of HOTAIR resulted in the loss ofH3K27 trimethylation and EZH2 binding but increased interaction of RNApol II with the promoter region, and this is consistent with cooperativePRC2/HOTAIR induction of GDF15. The effects of HOTAIR on GDF15 werefurther evaluated using two different siRNAs for HOTAIR in order tominimize possible off-target effects; both siHOTAIR I and II exhibited asimilar level of knockdown efficiency. Both siRNAs for HOTAIR decreasedluciferase activity in Panc1 cells transfected with constructscontaining the proximal −3500 to +41, −1086 to +41 and −474 to +41regions of the GDF15 promoter, whereas the effects of siHOTAIR wereminimal in Panc1 cells transfected with a construct containing only the−133 to +41 region of the promoter. These results suggest thatHOTAIR/PRC2 coordinately regulate GDF15 in pancreatic cancer cells; incontrast, this gene was not affected by HOTAIR overexpression inMDA-MB-231 cells, further demonstrating cell context-dependentdifferences in HOTAIR/PRC2-regulated genes.

As MiaPaCa2 and Panc28 cells express relatively low levels of HOTAIR,the functional and genomic effects of HOTAIR overexpression wereinvestigated and compared with overexpression of HOTAIR as previouslyreported in MDA-MB-231 cells and knockdown of HOTAIR in Panc1 and L3.6pLcells. HOTAIR overexpression enhanced MiaPaCa2 and Panc28 cell invasionin a Boyden chamber assay, and this was consistent with previousfunctional studies in breast and colon cancer cells. HOTAIRoverexpression decreased PCDBH5 and PCDH10 (as observed in MDA-MB-31cells) but not JAM2 in MiaPaCa2 cells, whereas in Panc28 cells PCDH10and JAM2 were induced and PCDBH5 was unchanged by HOTAIR overexpression.In contrast, overexpression of HOTAIR either decreased or did not affectLAMB3, ABL2, SNAI or LAMC2 mRNA levels in MiaPaCa2 and Panc28 cells,whereas these genes were all induced by HOTAIR overexpression inMDAMB-231 cells. The HOTAIR-repressed genes were also investigated byoverexpression of HOTAIR in MiaPaCa2 and Panc28 cells. HOTAIRdownregulated GDF15, MX1, IL28, IL28A, IL28B and IL29 only in MiaPaCa2cells and the remaining genes were unchanged in both cell lines.Overexpression of HOTAIR in MiaPaCa2 and Panc28 cells increased cellproliferation and the % of cells in S phase and decreased the % of G2/M(in Panc28 but not in MiaPaCa2 cells). Thus, results of both HOTAIRknockdown and overexpression in pancreatic cancer cells furtherdemonstrate differences between HOTAIR-regulated genes in pancreaticcells and breast cancer cells, and also show differences in HOTAIRfunction in MiaPaCa2 and Panc28 cells that express low levels of thisncRNA.

The functional effects of siHOTAIR in Panc1 and L3.6pL cells thatoverexpressed this ncRNA were further investigated. Transfection ofsiHOTAIR I and II inhibited Panc1 and L3.6pL growth, which wassignificant after 4 days, and 450% growth inhibition was observed after6 days (FIGS. 2A and 2B). Moreover, the siHOTAIR I and IIoligonucleotides also caused a measurable decreased expression of tworepresentative growth promoting genes (cyclin D1 and cyclin E),demonstrating a role for HOTAIR in expression (upregulation) of thesegenes in parts of the cell cycle (FIGS. 2C and 2D). Further, siHOTAIRscaused a G0/G1- to S-phase arrest in Panc1 cells (32 h), whereas inL3.6pL cells there was a decrease in the percent of cells in G0/G1 andincrease in G2/M, indicating pancreatic cancer cell context-dependentdifferences in the effects of HOTAIR on cell cycle progression (notshown). siHOTAIRs significantly decreased Panc1 and L3.6pL invasionusing a Boyden chamber assay (FIGS. 3A and 3B, respectively); siHOTAIRII also enhanced Annexin V staining associated with induction ofapoptosis in Panc1 and L3.6pL cells (FIG. 3C), and siHOTAIR also inducedPARP cleavage in both cell lines (data not shown). L3.6pL cellstransfected with siHOTAIR II were used in a mouse xenograft model, andup to 16 days after knockdown of HOTAIR by RNAi there was a dramaticdecrease in pancreatic tumor volume (FIG. 4A) and weight (FIG. 4B), andHOTAIR expression (FIG. 4C). Immunohistochemical staining of tumortissue indicated an increase in terminal deoxyribonucleotidetransferase-mediated nick-end labeling (TUNEL) staining and a decreasein proliferation markers (Ki67 and PCNA) in siHOTAIR vs.siCT-transfected tumors. These in vivo data complement the functional invitro studies of HOTAIR and confirm the pro-oncogenic activity of thislncRNA in pancreatic cancer cells and tumors.

Discussion:

HOTAIR was initially identified as one of the 231 ncRNAs associated withhuman HOX loci, and HOTAIR resided in the HOXC locus but repressedtranscription in the more distal HOXD locus in foreskin fibroblasts.HOTAIR interacted with the PRC2 complex and was required forPRC2-dependent histone H3 lysine 27 trimethylation and gene silencing.HOTAIRM1 and HO'TTIP are lncRNAs associated with the HOXA locus. SeeZhang et al. Blood 113(11):2526-34 (March 2009), and Wang et al. Nature472:120-124 (2011), respectively. Both of these lncRNAs differentiallymodulate gene expression in various cell and tissue types, butgenes/pathways modulated by these lncRNAs are PRC2-independent.

HOTAIR has also been characterized as a negative prognostic factor inbreast, liver and colon cancer patients, and the results of this studydemonstrate that HOTAIR is also overexpressed in human pancreatic tumorscompared with nontumor tissue (FIGS. 1A through 1C). Moreover, there isalso evidence that HOTAIR is more highly expressed in more aggressiveand invasive pancreatic tumors (FIGS. 1A through 1C). HOTAIR functionwas investigated in knockdown studies and indicates that this ncRNAenhances pancreatic cancer cell invasion, inhibits cell growth,modulates cell cycle progression and induces apoptosis in vitro, andHOTAIR knockdown in L3.6pL cells inhibited tumor growth in athymic nudemice bearing these cells as xenografts (FIGS. 4A-4C). The results ofHOTAIR overexpression in MiaCaPa2 cells and Panc28 cells were alsoconsistent with the pro-oncogenic activity of HOTAIR, although therewere some cell context-dependent differences. Thus, HOTAIR not only hasa role in invasion of pancreatic, breast, colon and liver cancer cells,but also exerts distinct pro-oncogenic activities in pancreatic cancerassociated with increased cell survival and proliferation, andrepression of interferon-related genes. This was further supported by asimilar result from GSEA analysis of Panc1 cells and human tumors,demonstrating that at least 50% of the gene sets were associated withcell cycle progression and proliferation.

Modulation of HOTAIR expression in breast and colon cancer cells, andtumors results in both enhanced and suppressed expression of genes, anda subset of genes repressed by HOTAIR in these cancer cells were alsocoregulated by PRC2. A comparison of the gene expression data modulatedby HOTAIR overexpression in MDA-MB-231 cells and HOTAIR knockdown inPanc1 cells showed some overlap in expression of individual genes;however, a heat map of induced/repressed genes illustrates significantdifferences between the cell lines. Moreover, only nine of the 854 genescoregulated by HOTAIR/PRC2 in MDA-MB-231 cells were also affected byHOTAIR knockdown in Panc1 cells. Among these genes, only OC1AD2 andRSAD2 were induced in Panc1 cells, whereas only minimal repression wasobserved after HOTAIR overexpression in MDA-MB-231 cells.

HOTAIR-dependent gene regulation in pancreatic cancer cells wasinvestigated here by HOTAIR knockdown in Panc1 and L3.6pL cells, andHOTAIR overexpression in Panc28 and MiaPaCa2 cells using a set of genesrepressed (JAM2, PCDH10 and PCDHB5) and induced (ABL2, SNAIL, LAMB3 andLAMC2) by HOTAIR overexpression in MDA-MB-231 cells. HOTAIR knockdown oroverexpression gave variable results among the four different pancreaticcancer cell lines, which in turn exhibited minimal overlap with respectto HOTAIR-dependent regulation of this set of genes in MDA-MB-231 cells.These results were consistent with the differences observed in the genearrays from Panc1 vs. MDA-MB-231 cells.

A second set of genes identified in the microarray that weresignificantly induced after HOTAIR knockdown were further investigatedin Panc1 and L3.6pL cells transfected with siHOTAIR, and all wereinduced in both cell lines. Previous studies show that all seven genesexhibit tumor suppressor-like activities, and four genes, namely IL29,IL28A, IL28B and IFTM1, were among a subset of severalinterferon-regulated genes suppressed by HOTAIR in Panc1 cells.Transfection of Panc1 and L3.6pL cells with siEZH2, a key component ofthe PRC2 complex, or siSuz12, another PRC2 component, showed that onlyGDF15 was coregulated by HOTAIR and EZH2 (PRC2), and ChIP assays incells transfected with siCT vs. siHOTAIR confirmed that GDF15 was aHOTAIR/PRC2-regulated gene. In contrast, the interferon-related geneswere not affected by EZH2 knockdown and the mechanisms ofPRC2-independent but HOTAIR-mediated suppression of these genes arecurrently being investigated. These observations are consistent withrecent reports showing that HOTAIR regulation of multiple genes isEZH2-independent. Consistent results were observed for theHOTAIR-regulated gene sets in pancreatic cancer patients and afterHOTAIR knockdown in pancreatic cancer cells, suggesting that knockdownof endogenous HOTAIR rather than overexpression can be preferable forinvestigating ncRNA-dependent gene regulation and function.

In summary, it is demonstrated in this description that HOTAIR is anegative prognostic factor for pancreatic cancer patients and exhibitspro-oncogenic activity in both in vitro and in vivo bioassays.HOTAIR-dependent gene regulation in pancreatic cancer cells is complexand differs significantly from a previous report in breast cancer cells.Nevertheless, HOTAIR knockdown in cells overexpressing this ncRNA gaveconsistent results using a subset of highly regulated genes, suggestingthat HOTAIR-mediated suppression of genes in pancreatic cancer is bothPRC2-dependent and PRC2-independent. Subsequent studies described hereinare focused on the mechanisms associated with suppression and activationof genes by HOTAIR in pancreatic cancer and development of therapeuticstrategies that target HOTAIR.

The following is a description of the investigation of the regulation oflncRNA by Specificity Protein Transcription Factors (SpTFs).

Introduction:

The negative prognostic significance and pro-oncogenic activity ofHOTAIR in pancreatic cancer is established in the study described above.Specifically, it is demonstrated above that HOTAIR repression of geneswas both PRC2-dependent and -independent. However, there was minimaloverlap between HOTAIR/PRC2-repressed genes in breast cancer cells vs.HOTAIR-repressed genes in Panc1 pancreatic cancer cells. Theanti-apoptotic gene GDF15 was identified as a HOTAIR/PRC2-regulated genein pancreatic cancer cells by knockdown of HOTAIR and PRC2 componentsEZH2 and SUZ12, whereas repression of the interferon-inducible proteinMx1 and interleukin-29 (IL-29) was HOTAIR-dependent but PRC-2independent.

Specificity protein 1 (Sp1) is a transcription factor (TF) that is alsooverexpressed pancreatic cancer and is a negative prognostic factor forpatient survival. Studies by the inventors have shown that Sp1, Sp3, andSp4 are highly expressed in pancreatic and other cancer cells and theseTFs play an important pro-oncogenic role in these cells due to theirregulation of Sp-dependent growth promoting (EGFR, c-MET, PTTG-1, cyclinD1), survival (bcl-2 and survivin), angiogenic (VEGF and VEGFR1/R2), andinflammatory (p65) genes. Moreover, knockdown of Sp1 in pancreatic andother cancer cells gives some of the same responses observed usingsiHOTAIR. See, e.g., FIG. 1D, FIGS. 2A-2D, and FIGS. 3A and 3B.Specificity protein transcription factors (SpTFs) are a prototypicalexample of “non-oncogene addiction” by cancer cells and their importanceas drug targets is due to the differential expression of Sp proteins incancer (high) vs. non-cancer (low) tissue, and this correlates withtheir decreased expression in animals and humans with age. The inventorshypothesized that HOTAIR was regulated by an SPTF and that agents thatdownregulate SpTFs might also downregulate HOTAIR.

Results and Discussion:

The similar downstream regulatory effects of Sp1 and HOTAIR werecharacterized to establish a potential connection between Sp1 andHOTAIR. Sp1 knockdown surprisingly revealed that in Panc1 cellstransfected with iSp1 oligonucleotide (Sp1-KO) more genes were inducedthan repressed, suggesting that Sp1, like HOTAIR, was also involved ingene induction. Moreover, it was observed that 377 genes were commonlyregulated by HOTAIR and Sp1 (332 repressed, 45 activated). Some of theeffects of iSp1 can be due, in part, to decreased expression of HOTAIR.Independent knockdown of both HOTAIR and Sp1 increased expression ofGDF15 (PRC2-dependent) and the PRC2-independent Mx1 and IL-29 genes.FIGS. 5A-5C. For the same three genes, a chromatin immunoprecipitation(ChIP) assay demonstrated that knockdown of HOTAIR and Sp1 enhancedPOLII binding to their respective gene promoters, and these data wereconsistent with the enhanced gene expression data (FIGS. 5A-5C). A ChIPassay was also used to determine Sp1 and EZH2 binding to the −1618 to−1267 (GDF15), −527 to −299 (Mx1), and −585 to −205 (IL-29) region ofthese genes. Because the vast majority of PRC2 bound genomic sequencesare localized within 1 kb of a transcription start site, the multipleprimer sets were designed for detecting this genomic region of targetgene promoters. Results of the ChIP assay confirmed that Sp1 and EZH2bound to the GDF15 promoter, indicating that Sp1 and possibly other Spproteins can form part of the PRC-2/HOTAIR complex that is required forgene repression. This is the first demonstration of the novel repressivefunction of Sp1 in combination with PRC2 and HOTAIR. Furthermore, IL-29and Mx1 regulation was HOTAIR/Sp1-dependent but PRC-independent, whichis also the first demonstration of this novel repressor complex.

Comparable procedures and arrays to determine common HOTAIR-Sp3 andHOTAIR-Sp4 regulated genes. Using knockdown assays, it was observed that209 genes were commonly regulated by HOTAIR and Sp3 (75 repressed, 134activated). Similarly, it was observed that 208 genes were commonlyregulated by HOTAIR and Sp4 (83 repressed, 124 activated). The resultssuggested that both Sp3 and Sp4 also cooperatively interacted withHOTAIR to repress genes.

The above results demonstrate that HOTAIR and Sp1 coordinately decreaseexpression of several “tumor-suppressor-like” genes. Moreover, knockdownof Sp proteins by RNAi gives some of the same responses includinginhibition of cell growth, induction of apoptosis and inhibition ofmigration/invasion. Therefore, it was investigated whether SpTFs alsoregulate HOTAIR expression in cancer cells.

Using short interfering RNAs corresponding to SpTF genes for RNAiassays, expression of the SpTF were successfully knocked down. Sp1 wasknocked down using an oligonucleotide with a sequence set forth hereinas SEQ ID NO:19. Sp3 was knocked down using an oligonucleotide with asequence set forth herein as SEQ ID NO:20. Sp4 was knocked down using anoligonucleotide with a sequence set forth herein as SEQ ID NO:21.Results in FIG. 6A show that siSp1, siSp3, and siSp4 decreased Sp1, Sp3,and Sp4 mRNA levels in Panc1 cells compared to siCT (control).Comparable effects were observed for Sp proteins (data not shown).Moreover, the application of siSp1, siSp3 and siSp4 surprisinglyresulted in a significant decrease in the expression of HOTAIR and twoother HOX region ncRNAs (HOTAIRM1 and HOTTIP). FIG. 6B. In this assay,HOTAIRM was amplified and detected using forward and reverseoligonucleotide sequences set forth herein as SEQ ID NOS:3 and 4,respectively. Additionally, HOTTIP was amplified and detected usingforward and reverse oligonucleotide sequences set forth herein as SEQ IDNOS:10 and 11, respectively. Examination of the HOTAIR promoter showedthat there are at least two GC-rich Sp binding sites. A ChIP assay usingprimers that specifically target the GC-rich region (Primer II) andnon-GC-rich regions (Primers I and III) showed that Sp1 preferentiallybound the GC-rich region (Primer II) (not shown). These resultsdemonstrate for the first time that expression of HOTAIR and otherlncRNAs are regulated, at least in part, by SpTFs and opens thepossibility that anticancer drugs that downregulate Sp1, Sp3, and Sp4 incancer cells and tumors will target HOTAIR and other Sp-regulatedlncRNAs.

The following is a description of an analysis of compounds targetingHOTAIR through downregulation of SpTFs.

Introduction:

Sp transcription factors (e.g., Sp1, Sp3, and Sp4) are highly expressedin tumor vs. non-tumor tissues. Sp1 is a negative prognostic factor forpancreatic cancer survival, and this correlates with the prognosticsignificance of HOTAIR, as demonstrated above. The high pro-oncogenicactivity of SpTFs coupled with the “addiction” of pancreatic cancers forthese TFs makes them ideal drug targets, which is a major focus of theinventors. Drug-induced downregulation of SpTFs in pancreatic and othercancer cell lines is proteasome-dependent (minor) or -independent(major), and the specific pathways are both drug- and cellcontext-dependent. Compounds that regulate SpTFs were investigated fortheir effect on HOTAIR expression.

Results and Discussion:

Synthetic triterpenoids CDODA-Me (methyl2-cyano-3,11-dioxo-18-olean-1,12-dien-30-oate) and the CF₃ analogCF₃DODA-Me downregulate Sp1, Sp3, and Sp4 proteins in multiple cancerlines. Previous studies showed that the synthetic triterpenoid CDODA-Medecreased microRNA (miR)-27a and increased ZBTB10, an SpTF repressor incolon cancer cells. Similarly, CF₃DODA-Me and CDODA-Me downregulate Sp1,Sp3, and Sp4 proteins in Panc1 cells. FIG. 7A. Similar results wereobserved other pancreatic cancer cell lines (data not shown). FIG. 7Bdemonstrates that treatment of CDODA-Me and CF3DODA-Me also decreasedHOTAIR expression in Panc1 cells, which represents the first example ofdrug-induced downregulation of an oncogenic lncRNA. It is noted thatHOTAIR was decreased by both the triterpenoids and by Sp knockdown by>50%.

Previous studies with CDODA-Me and the structurally-related triterpenoidanalog methyl-2-cyano-3,12-dioxooleana-1,9-dien-28-oate (CDDO-Me) showedthat SpTF downregulation in colon and pancreatic cancer cells,respectively, was due to repression of miR-27a and induction of ZBTB10,an Sp transcriptional repressor that is regulated by miR-27a. Moreover,in pancreatic cancer cells, drug-mediated Sp protein and miR-27adownregulation and induction of ZBTB10 was due to induction of reactiveoxygen species (ROS), and the process was inhibited by cotreatment withantioxidants such as DTT or GSH. Similar results have been observed forother anticancer agents such as betulinic acid and GT-094 (a NO-NSAID)in colon and celastrol in bladder cancer cells. In this study, theeffects of CDODA-Me and CF₃DODA-Me on these targets were investigated inPanc1 cells. Results illustrated in FIGS. 8A-8C show that CDODA-Me- andCF₃DODA-Me-mediated induction of ZBTB10, downregulation of miR-27a, andrepression of Sp1, Sp3, and Sp4 was inhibited after cotreatment withantioxidants DTT or GSH. This suggests that drug-induced ROS can alsorepress HOTAIR. Further, FIG. 81) shows that CF₃DODA-Me-inducedrepression of HOTAIR is also attenuated by GSH. Moreover, the fact thatCDODA-Me decreased Sp1, Sp3, and Sp4 in pancreatic cancer cells (FIG.8C) demonstrates that agents such as CDODA-Me that downregulate Sp1,Sp3, and Sp4 also decrease HOTAIR, an Sp-regulated gene. Moreover, wehave recently demonstrated that miR-20a and miR-17-Sp regulateexpression of a second Sp-repressor, ZBTB4, and ROS also disruptsmiR-20a/17-5p-ZBTB4, and thus, also likely influence expression ofHOTAIR and other pro-oncogenic lncRNAs.

In another instance, tolfenamic acid (TA), an NSAID that also decreasesSp proteins in cancer cells (J. Nat'l Cancer Inst. 98:855 (2006); CancerRes. 67:3286 (2007); Mol. Cancer. Ther. 8:533 (2009)), decreased HOTAIR,cancer cell growth, and expression of the Sp-regulated genes EZH2,cyclin D1 and cyclin E in MDA-MB-231 breast cancer cells. FIG. 9A-9C.

These results clearly demonstrate that drugs inducing Sp downregulationalso target HOTAIR, an Sp-regulated oncogenic ncRNA.

The following is a description of further characterizations of HOTAIRregulation by SpTFs and the implications for drug targeting.

Introduction:

LncRNA expression is polII-dependent. However, the trans-acting factorsthat regulate these RNAs are essentially unknown. Results describedhereinabove demonstrate that knockdown of Sp proteins by RNAi results indecreased HOTAIR expression (see, e.g., FIG. 6B), and ChIP analysis forSp1 demonstrates that this protein binds the GC-rich region of theHOTAIR promoter. Thus, the high expression and “addiction” of Panc1cells to Sp1, Sp3, and Sp4 transcription factors results in high levelsof HOTAIR, an Sp-regulated gene and this will be further investigated inthe panel of pancreatic cancer cells will variable HOTAIR expression. InPanc1 and other cancer cells, overexpression of Sp transcription factorsis related to miR-27a-dependent repression of ZBTB10 andmiR-20a/miR-17-5p-dependent repression of ZBTB4. Both ZBTB10 and ZBTB4are transcriptional or “Sp” repressors that bind GC-rich element andinactivate gene expression. Investigations into the regulation of HOTAIRby miR-27a/miR-20a/17-5p-ZBTB10/ZBTB4-Sp protein circuits are describedbelow.

Aim A:

Investigate Sp TFs Regulation of HOTAIR Expression in Pancreatic CancerCells.

(i) RNA interference studies. The HOTAIR-overexpressing (Panc1, L3.6pL)cells are transfected with siRNAs for Sp1 (siSp1), Sp3 (siSp3), Sp4(siSp4) and their combination (siSp1/3/4), as previously described andeffects on HOTAIR are determined by real time PCR. A similar approachfor pancreatic cancer cells that express lower levels of HOTAIR (e.g.,BxPC3, MiaPaCa-2 and Panc28) is also used because this will determine ifother factors or lower levels of Sp1, Sp3, and Sp4 are responsible fordecreased expression of HOTAIR. Overexpression of Sp proteins in cellsexpressing low levels of HOTAIR are also determine if limiting levels ofSp1, Sp3, and Sp4 influence HOTAIR expression and function.

(ii) ChIP assay of Sp binding to HOTAIR promoter. The binding of Sp1,Sp3, and Sp4 to the HOTAIR promoter is determined to confirm theoccupation of these GC-rich sites by individual Sp transcription factorsin pancreatic cancer cells and the loss of these proteins after RNAi(i.e., knockdown of Sp1, Sp3, and Sp4).

Aim B:

Investigate Sp-dependent regulation of HOTAIR by miR-27a:ZBTB10 andmiR-20a/17-5p:ZBTB4, miR-27a and miR-20a/miR-17-5p are highly expressedin many cancer cell lines, and studies in several cancer cell linesdemonstrated that high levels of Sp1, Sp3, and Sp4 in these cells wasdue, in part, to miR-27a-dependent inhibition of the transcriptionalrepressor ZBTB10 and miR-20a/17-Sp inhibition of ZBTB4. ZBTB10 and ZBTB4bind and partially displace Sp1, Sp3, and Sp4 from GC-rich elements toinhibit gene expression. The inventors have shown that antagomirs ofmiR-27a, miR-20a and miR-17-5p with ZBTB4 or ZBTB10 overexpressionresulted in downregulation of Sp1, Sp3, Sp4, and Sp-regulated genes inbreast and pancreatic cancer cells. Therefore, HOTAIR expression inpancreatic cancer cells is likely regulated by miR-27a:ZBTB10-HOTAIR andmiR-20a/miR-17-5p:ZBTB4-HOTAIR crosstalk.

(i) Effects of antagomiR-27a and ZBTB10 on HOTAIR expression. Panc1 andL3.6pL are transfected with as-miR-27a or ZBTB10 expression, and HOTAIRexpression is determined by real time PCR, and expression of Sp1, Sp3,and Sp4 proteins are determined by western blots. Different amounts oftransfected ZBTB10 or as-miR-27a and different treatment times are usedto maximize responses using approaches previously described.

(ii) Effects of antagomiR-20a, antagomiR-17-5p and ZBTB4 on HOTAIRexpression. The dose-dependent effects of the antagomirs or ZBTB4overexpression on HOTAIR expression, and Sp1, Sp3, and Sp4 proteins aredetermined as described above in Aim B(i).

(iii) ChIP assay for ZBTB0 and ZBTB4. ChIP assays are used determineoccupation of the GC-rich elements of HOTAIR by Sp1, Sp3, Sp4, ZBTB10,ZBTB4 and polII in pancreatic cancer cells transfected with theantagomirs, ZBTB10 and ZBTB4 expression plasmids. Based on results ofthe studies described herein, loss of Sp1, Sp3, Sp4 and polIIinteractions and increased recruitment of ZBTB100 and ZBTB4 to theGC-rich sites are expected.

Expected Results and Alternative Approaches.

The investigations described herein will confirm the critical roles ofSp1, Sp3, and Sp4 in regulating HOTAIR in pancreatic cancer cells andalso demonstrate that, because regulation of Sp proteins is dependent onmiR-27a and miR-20a/17-5p suppression of ZBTB10 and ZBTB4 (29, 30, 38,50, 51), HOTAIR expression is also regulated by miR-27a:ZBTB10.Mithramycin is a drug that binds GC-rich sites and downregulates Sp1 andother Sp-regulated genes. This compound will also be used in thisExample to directly target HOTAIR, and a decrease in HOTAIR expressionand displacement of Sp protein from the GC-rich HOTAIR promoter isexpected, whereas recruitment of ZBTB4 or ZBTB10 should not be observed.

The following is a description of further characterizations ofdrug-induced repression of HOTAIR.

Introduction:

The inventors have demonstrated that several different anticancer agentsdecrease Sp1, Sp3, and Sp4 protein expression in cancer cell linesthrough multiple mechanisms which are both drug- and cellcontext-dependent. SpTFs are excellent drug targets due to theiroverexpression in tumors and relatively low expression in normal tissuewhich is consistent with the reported decrease in Sp1 levels withincreasing age. Data described hereinabove demonstrate that bothCDODA-Me and CF3DODA-Me decrease HOTAIR, downregulate Sp proteins andmiR-27a, and induce ZBTB10 expression in pancreatic cancer cells (see,e.g., FIGS. 7A-7B and 8A-8D). In this description, investigations aredescribed that will demonstrate that CDODA-Me and CF3DODA-Me decreaseHOTAIR through ROS-dependent perturbation of miR-27a:ZBTB10, whichresults in downregulation of Sp1, Sp3, Sp4, and HOTAIR. In addition, theinventors have recently demonstrated that other ROS-inducing anticanceragents disrupt miR-20a/miR-17-5p-ZBTB4 interactions. Accordingly, therole of this pathway on the effects of CDODA-Me and CF3DODA-Me on HOTAIRexpression will also be determined.

Aim A:

CDODA-Me/CF3DODA-Me Downregulate HOTAIR in Pancreatic Cancer Cells.

(i) HOTAIR downregulation. Panc1 and other HOTAIR (high) expressing celllines are treated with 1.0, 2.5, 5.0, and 7.5 μM CDODA-Me or CF3DODA-Mefor 6, 12, 18, 24, and 48 hr. HOTAIR levels are determined by realtime-PCR. Using optimal concentrations and times, the effects of thesecompounds on HOTAIR-induced or -repressed genes are determined tofurther confirm that these compounds down-regulate HOTAIR.

(ii) Role of ROS and ROS-dependent perturbation of the miR-27a:ZBTB10-Spand miR-20a/17-5p:ZBTB4-Sp pathways. Using the same treatment protocolindicated above, the effects of CDODA-Me/CF3DODA-Me on ROS, miR-27a,ZBTB10, Sp1, Sp3, and Sp4 levels are determined as described, and theeffects of antioxidants (catalase, DTT, GSH, NAC) on this pathway andHOTAIR expression are also determined. Using an optimal treatment timeand concentration of CDODA-Me and CF3DODA-Me, cells are transfected withmiR-27a or miR-20a mimics or siZBTB10/siZBTB4 (knockdown) to reverse thedrug-induced downregulation of HOTAIR and confirm the role ofROS-MiR:ZBTB in mediating the repression of HOTAIR. In addition, theeffects of the treatments on interaction of Sp proteins and ZBTB10 andZBTB4 with the GC-rich HOTAIR promoter are determined in a ChIP assay.

(iii) Role of c-Myc in ROS-dependent repression of HOTAIR. The proposedinvestigations coupled with the established results support thehypothesized linkage between drug-induced ROS and ROS-mediated effectson miR-ZBTB which results in repression of Sp-TFs and HOTAIR. A recentstudy demonstrated that ROS(H₂O₂) induces chromatin shifts andrelocalizes methyl transferases/PRC4 from non-GC-rich to GC-rich areas.One of the repressed genes is Myc, which also regulates miR-27a andmiR-20a/17-5p (miR-17-92 cluster). FIG. 10 is a western blotdemonstrating that CF3DODA-Me downregulates Myc protein expression inPanc1 cells. Comparable responses for other ROS inducers have also beenobserved (data not shown). Therefore, in this investigation, Mycexpression is knocked down by RNAi (siMyc) and the effects onmiR:ZBTB-Sp axis and HOTAIR expression are determined. A ChIP assay onthe HOTAIR promoter is used to confirm this pathway. It is possible thatROS-induced relocalization of repressor complexes such as PRC4 willdirectly affect HOTAIR transcription. In this scenario, a ChIP assay isused to determine changes in binding of PolII, methylated histones andPRC4 complex members on the GC-rich HOTAIR promoter.

Aim B:

CDODA-Me/CF3DODA-Me decrease HOTAIR and tumor growth in vivo. Knockdownof HOTAIR in L3.6pL cells by RNAi dramatically decreased tumor growth ina nude mouse xenograft model. In this Aim, stably transfected L3.6pLcells are used in an orthotopic model of pancreatic cancer which alsoundergoes liver metastasis. Mithramycin blocks Sp-mediatedtransactivation and, thus, is used as a third “control” drug thattargets Sp proteins and should also decrease HOTAIR expression.

(i) Role of HOTAIR in pancreatic tumor growth and metastasis. L3.6pLcells are stably transfected with a lentiviral-shRNA to knockdown HOTAIRand the effects of wild-type L3.6pL cells (transfected with anon-specific lentiviral shRNA) vs. HOTAIR knockdown cells areinvestigated in an orthotopic pancreatic tumor model (see below).Pancreatic cancer cells that express low levels of HOTAIR (e.g., BxPC3or MiaPaCa2) are transfected with a lentiviral expression vector forHOTAIR (or a control) and the effects of variable HOTAIR expression aredetermined in a xenograft model (these cells do not form tumors in theorthotopic model).

(ii) Animal treatment. Male athymic nude mice can be obtained fromcommercial sources and their use must be approved by an InstitutionalAnimal Care and Use Committee. The mice are housed under specificconditions and in facilities approved by the American Association forAccreditation of Laboratory Animal Care. Ten animals are used for eachtreatment group. Panc1 and Panc28 cells are used in the xenograft study.Cells are harvested by exposure to trypsin and resuspended in serum-freeHanks' balanced salt solution (HBSS). Viability is assessed by trypanblue exclusion, and only single-cell suspensions exhibiting greater than95% viability will be used. For subcutaneous tumors, tumor cells (1×10⁶cells) suspended in a volume of 200 μL are implanted subcutaneously inthe flank of nude male animals using a 27-gauge needle. Tumors areallowed to grow unperturbed for 10-14 d and when palpable tumors (200mm³) first appear, mice are randomly assigned to treatment or controlgroups. Mice are treated (10 per treatment group) with placebo orCF3DODA-Me, CDODA-Me (2, 10, or 20 mg/kg/d), or Mithramycin (0.1 and 0.2mg/kg/d) (in corn oil) administered every second day for 4 to 6 weeks(depending on appearance and size of control tumors). L3.6pL cells areused for orthotopic pancreatic tumor studies. Cells are injecteddirectly into the pancreas and tumors are analyzed as previouslydescribed. Body, organ and tumor weights are determined and tissues areused for histopathology. Metastasis to the liver and other organs ismeasured according to known methods.

(iii) Analysis of tumor tissues. Tumor sections from animals will beprepared for in situ hybridization and immunohistochemical analysis ofproteins and in situ hybridization for Sp and Sp-regulated gene productsand mRNAs. Western blot analysis of Sp and Sp-regulated genes will bedetermined using tumor lysates. RNA extracted from control and treatedtumors will be used to determine HOTAIR, ZBTB10, ZBTB4, miR-27a,miR-20a, miR-17-5p, and HOTAIR-regulated genes identified in Aim 1.

Expected Results and Alternative Approaches.

Aims A and B will provide complementary in vitro and in vivo results anddemonstrate that CDODA-Me/CF3DODA-Me decrease HOTAIR throughperturbation of miR-27a:ZBTB10 and miR020a/17-5p:ZBTB4, which results inSp downregulation (see, e.g., FIGS. 7A and 8C). In addition, HOTAIRstably transfected (or knockdown) cells in orthotopic or xenograftmodels can be used. Mithramycin is useful as an alternative model for adrug that inhibits Sp-dependent transactivation. Tolfenamic acid alsodecreases Sp proteins in Panc1 cells through activation of proteasomes,which presents an additional pathway for investigation as to the role inregulating HOTAIR. Finally, transgenic animal models for pancreaticcancer that express endogenous mutant Kras and p53 alleles can bedeveloped in the laboratory for in vivo studies on HOTAIR and lncRNAregulation.

The following is a description of the regulation of MALAT-1 (lncRNA) bySpTFs.

Introduction:

It is demonstrated hereinabove that HOTAIR, a lncRNA, is regulated bySpTFs, and that compositions that suppress SpTF expression also suppressHOTAIR expression. Therefore, the inventors investigated the regulationof other lncRNAs by SpTFs.

Metastasis-Associated-in-Lung-Adenocarcinoma-Transcript-1 (MALAT-1)(Yang et al. Cell 147(4):773-88 (November 2011)) is an lncRNA that isoverexpressed in multiple cancer cell lines and tumors (Huarte, M., andRinn, J. L., “Large Non-Coding RNAs: Missing Links in Cancer?” Hum. Mol.Genet. 19:R152-161 (2010); Perez, D. S., et al., “Long, AbundantlyExpressed Non-Coding Transcripts Are Altered in Cancer,” Hum. Mol.Genet. 17:642-655 (2008); Li, L., et al., “Role of Human Noncoding RNAsIn the Control of Tumorigenesis,” Proc. Nat'l Acad. Sci. USA106:12956-12961 (2009)), and MALAT-1 expression is a prognostic factorfor decreased survival of stage 1 non-small cell lung cancer (NSCLC)(Ji, P., et al., “MALAT-1, a Novel Noncoding RNA, and Thymosin Beta4Predict Metastasis and Survival in Early-Stage Non-Small Cell LungCancer,” Oncogene 22:8031-8041 (2003)). MALAT-1 expression is alsoassociated with metastasis in NSCLC patients and MALAT-1 expression iscorrelated with poor prognosis (survival/recurrent/metastasis) insquamous cell carcinoma of the lung, hepatocellular carcinoma, bladderand colorectal cancer. Moreover, functional studies determined byMALAT-1 knockdown or overexpression indicate that MALAT-1 enhances celland tumor growth, migration, invasion and epithelial-to-mesenchymaltransition. MALAT-1 mechanisms of action include (a) acting as a decoyto bind the tumor suppressor PSF; (b) localizing in nuclear speckles toregulate RNA posttranscriptional modifications; (c) regulating genesinvolved in synapse function; and (d) regulating growth promoting genesthrough interaction with polycomb protein 2 (Pc2/CBX4) to promote E2F1sumoylation and enhance expression of growth promoting genes. Thus,MALAT-1 exhibits a broad range of activities in different cell lines.MALAT-1 expression is enhanced in human tumors vs. non-tumor tissues inseveral cancers including pancreatic cancer; moreover, human MALAT-1 ishighly conserved in mice (hepcarin/hcn) and other mammals (unlikeHOTAIR) and its function can be investigated in mouse models.

The results described herein confirm that MALAT-1 exhibits pro-oncogenicactivity in pancreatic cancer cells through regulation of multiple genesand pathways. Moreover, like HOTAIR, the present RNAi studies also showthat MALAT-1 expression is regulated by specificity protein (Sp)transcription factors in pancreatic cancer cells. Therefore, it ishypothesized that MALAT-1 is pro-oncogenic in pancreatic cancer cellsand in animal models and that MALAT-1 expression is regulated byspecificity protein (Sp) transcription factors and can be targeted byanticancer agents that downregulate Sp proteins.

Results:

MALAT-1 Expression in Pancreatic Cancer Cells.

Expression studies in various pancreatic cell lines demonstrated thatMALAT-1 is expressed in pancreatic cancer cells FIG. 11. MALAT-1 RNA wasdetected using forward and reverse oligonucleotides with sequences setforth herein as SEQ ID NOS:7 and 8. Furthermore, MALAT-1 can be detectedin serum obtained from pancreatic cancer patients (not shown) and thiscorrelates with the reported high expression of MALAT-1 in pancreatictumors compared to non-tumor tissues. Subsequent MALAT-1 knockdownstudies with RNA interference were conducted using two oligonucleotides(siMALAT-1a and siMALAT-1b), the sequences of which are set forth hereinas SEQ ID NOS:5 and 6, respectively. FIGS. 12A and 12B demonstrate thatloss of MALAT-1 in Panc1 and Panc28 cells, respectively, resulted insignificant inhibition of cell proliferation compared to cellstransfected with a control oligonucleotide (siLamin). Thus, MALAT-1 andMALAT-1-regulated genes play a role in pancreatic cancer cell growth.MALAT-1 knockdown by RNAi also significantly decreased transwellmigration in Panc28 and Panc1 cells as determined by a Boyden chamberassay (not shown). Additional studies on the role of MALAT-1 in cellsurvival and kinase signaling were performed, because both pathways areaffected, in part, by overexpression of growth factors and activatingRAS mutations. These aspects are common in many pancreatic cancer celllines and tumors. Results in FIGS. 13A and 13B demonstrate that MALAT-1knockdown induced PARP cleavage (a marker for apoptosis) and inhibitedAKT/MAPK (phosphorylated and total proteins, respectively) in Panc28cells. Similar results were observed in Panc1 cells (not shown).

Because MALAT-1 and MALAT-1-regulated genes are shown to play criticalroles in growth, survival and migration of pancreatic cancer cells,further confirmation and characterization will be performed in a panelof pancreatic cancer cell lines, which exhibit variable expression ofMALAT-1, as indicated in FIG. 11. This effort will identify both highand low expressors. HPDE cells (non-transformed) will serve as controlpancreatic tissue which has relatively low expression of MALAT-1.Additionally, a pancreatic cancer tissue array from the National DiseaseResearch Interchange will be used to investigate expression of MALAT-1in patient tissues. These fixed tissues will be used to examine MALAT-1expression by RNA in situ hybridization. Since HOTAIR is also highlyexpressed in pancreatic cancer cells and tissues, the HOTAIR antisensereverse and antisense forward primers that have previously beendescribed for in situ hybridization of fixed tumor tissue (Chisholm, K.M., et al., “Detection of Long Non-Coding RNA in Archival Tissue:Correlation With Polycomb Protein Expression in Primary and MetastaticBreast Carcinoma,” PLoS One 7:e47998, 2012, incorporated herein byreference in its entirety). Probes for MALAT-1 (400-500 nt) will bedeveloped based upon unique non-conserved sequences essentially asdescribed (Chintharlapalli, S., et al., “Inhibition of PituitaryTumor-Transforming Gene-1 in Thyroid Cancer Cells by Drugs That DecreaseSpecificity Proteins,” Mol. Carcinog. 50:655-667, 2011, PMCID:PMC3128656, incorporated herein in its entirety). Staining intensitieswill be scored using a grading system as reported for Rhabdomyosarcomaimmunostaining (Chadalapaka, G C., et al., “Inhibition ofRhabdomyosarcoma Cell and Tumor Growth by Targeting Specificity Protein(Sp) Transcription Factors,” Int. J. Cancer 132:795-806, 2013, PMCII):PMC3527649, incorporated herein in its entirety); comparisons of MALAT-1with HOTAIR and a control mRNA (GAPDH) will also be performed.

At least two high expressing and low expressing cell lines can be usedfor knockdown and overexpression of MALAT-1, respectively, to study thefunction(s) of this lncRNA in pancreatic cancer cells. Exemplaryinvestigation approaches are described here. A non-specificoligonucleotide (RNAi) or empty vector (expression) can be used ascontrols in all transfection studies.

The antiproliferative effects resulting from MALAT-1 knockdown can bedetermined in pancreatic cells in proliferation andanchorage-independent growth assays and by FACS analyses. Pancreaticcancer cells are transfected with siMALAT-1 accordingly to standardprotocols and the effects on cancer cell growth are determined after 1,2, 4, and 6 days by cell counting and the MTT assay. The effects ofsiMALAT-1 on anchorage-independent growth and cell cycle progression aredetermined and are routinely carried out in the laboratory. The effectsof MALAT-1 overexpression on cell proliferation, anchorage-independentgrowth and cell cycle progression are determined in at least 2 celllines that express low levels of MALAT-1.

siMALAT-1 or MALAT-1 overexpression can be used on high and lowMALAT-1-expressing cells as described above and their effects onapoptosis, activation of caspases 3, 8, and 9 (cleavage), PARP cleavage,TUNEL assay and DNA laddering by enhanced annexin V staining andreversal of these responses by caspase inhibitors (Z-VAD-fmk andLEHD-CHO) (routinely carried out in this laboratory) can be determined.If cells with low expression of MALAT-1 exhibit low endogenousapoptosis, the prosurvival activity of MALAT-1 overexpression can bedetermined in cells pretreated with an apoptosis-inducing agent such asTPA, and reversal of apoptosis by overexpressing MALAT-1 is determinedusing the apoptosis assays indicated above. In addition, induction ofchemo-resistance by MALAT-1 overexpression is determined against otherchemotherapeutic drugs including gemcitabine, doxorubicin andcis-platin.

The effect of MALAT-1 on cell migration and invasion characteristics canbe assessed. Pancreatic cancer cells expressing high or low levels ofMALAT-1 are transfected with siMALAT-1 or MALAT-1 expression vectors,respectively, and after 78 or 96 hr, cell migration and invasion aredetermined and quantitated in scratch and Boyden chamber assaysessentially, as previously described.

Previous reports suggest that MALAT-1 can play a role inepithelial-to-mesenchymal transition (EMT), which has been linked tocancer progression and metastasis. Cells that exhibit some mesenchymalmorphology and this cell line can be transfected with siMALAT-1 andexamined for changes in cell morphology and changes in expression of keydiagnostic genes associated with the reversal of EMT (e.g., β-catenin,N-cadherin, ZEB-1, ZEB-2, Snail, Slug, and related genes). Cellsexhibiting a more “epithelial-like” phenotype can be transfected withMALAT-1 expression plasmid and examined for phenotypic and genotypicchanges associated with EMT.

The described investigations are expected to further characterizeMALAT-1 as a key pro-oncogenic factor in pancreatic cancer cells andalso confirm overexpression in pancreatic tumor vs. non-tumor tissue. Inaddition to a tumor array, analysis can also proceed on pancreatic tumorsamples obtained from a study on metformin, which will add to results ofthe proposed research.

Regulation of MALAT-1 Expression by SpTFs.

As described above, Sp1, Sp3, and Sp4 TFs are overexpressed inpancreatic and other cancer cell lines, and Sp1 is a negative prognosticfactor for pancreatic cancer patient survival. SpTFs regulate expressionof genes associated with cell proliferation, survival, metastasis andinflammation, and several growth factor receptor tyrosine kinases. SpTFsare prototypical examples of non-oncogene addiction by cancer cells andare ideally suited as targets for mechanism-based drugs for thefollowing reasons: Sp 1 expression decreases with age in human androdent tissues; Sp1, Sp3, and Sp4 are highly expressed in cancer cellsand tumors compared to non-tumor tissues; and, many drugs that targetSpTFs do so through mechanisms/pathways that are highly upregulated incancer cells compared to non-cancer tissues, which facilitates drugspecificity (for cancer cells) and decreased toxicity to normal cells.Silencing of SpTFs and other in pancreatic cancer cells results ingrowth inhibition, apoptosis and decreased cell migration. These effectsare similar to those observed after knockdown of MALAT-1 in these samecell lines (see, e.g., FIGS. 12A, 12B, 13A, and 13B).

Accordingly, MALAT-1 was investigated further for possible regulation bySpTFs in pancreatic cancer cells, and to establish the possibility ofmanipulation by anticancer drugs that downregulate SpTFs. In an RNAistudy, Panc1 cells were exposed to a combination of siSp1/3/4 and tosiCT. FIG. 14 demonstrates that silencing of Sp1/3/4 (combined) by RNAiin Panc1 cells significantly decreased MALAT-1 expression, which isconsistent with the results described above for HOTAIR.

The regulation of MALAT-1 by SpTFs can be further characterized.Exemplary RNAi and ChIP assays are described. The MALAT-1 overexpressingpancreatic cancer cells are transfected with siRNAs for Sp1 (siSp1), Sp3(siSp3), Sp4 (siSp4) and their combination (siSp1/3/4), as previouslydescribed above, and effects on MALAT-1 expression are determined byreal time PCR. A similar approach for pancreatic cancer cells thatexpress lower levels of MALAT-1, as described above, because this willdetermine if other factors or lower levels of Sp1, Sp3, and Sp4 areresponsible for decreased expression of MALAT-1. Overexpression of Spproteins in cells expressing low levels of MALAT-1 is also determined iflimiting levels of Sp1, Sp3, and Sp4 influence MALAT-1 expression andfunction. ChIP assays are used to characterize the binding of Sp1, Sp3,and Sp4 to the MALAT-1 promoter and to confirm the occupation of GC-richsites by individual Sp transcription factors in pancreatic cancer cellsand the loss of these proteins after RNAi (i.e., knockdown of Sp1, Sp3,and Sp4).

Compounds that downregulate SpTFs also decrease MALAT-1 expression inpancreatic cancer cells.

As described above, the mechanisms of drug-induced downregulation ofSpTFs are due to activation of transcriptional and degradation(non-transcriptional) pathways, which are tumor type-, cell context-,and drug-dependent. Among the most effective agents that downregulateSpTFs in pancreatic cancer cells are the synthetic triterpenoidanticancer agents methyl 2-cyano-3,12-dioxooleana-1,9-dien-28-oate(CDDO-Me, bartoxolone) and methyl2-cyano-3,11-dioxooleana-1,12-dien-30-oate (CDODA-Me). Both compounds,as well as the potent 2-CF₃ analog of CDODA-Me (CF₃DODA-Me), activateROS in pancreatic cancer cells, which results in downregulation ofmiR-27a and induction of the Sp-repressor ZBTB10. Moreover, in some celllines the inventors have observed induction of a second Sp-repressor(ZBTB4) resulting from the downregulation of miR-20a/miR-17-5p.

Accordingly, the inventors investigated the possible regulation ofMALAT-1 by drugs known to suppress SpTFs. Results in FIG. 15A show thatdoses of 5.0 and 7.0 μM CDODA-Me and CF₃DODA-Me significantlydownregulate MALAT-1 expression in pancreatic Panc1 and L3.6pL cancercells. Additionally, both compounds were observed to induce ROS (datanot shown). Treatment of Panc1 cells with CF₃DODA-Me in the presence orabsence of glutathione (GSH) shows that the antioxidant inhibitsCF₃DODA-Me-induced downregulation of MALAT-1 (FIG. 15B), which isconsistent with involvement of the ROS pathway. As described above,CF₃DODA-Me-mediated downregulation of Sp1, Sp3, and Sp4 is alsoinhibited by cotreatment with GSH (FIG. 8C), implying involvement of thesame pathway.

Further investigations will provide additional insight to the regulatoryrelationship between SpTFs and MALAT-1. Exemplary studies are described.To study MALAT-1 downregulation, Panc1 and other pancreatic cancer celllines are treated with 1.0, 2.5, 5.0, and 7.5 μM CDODA-Me or CF₃DODA-Mefor 6, 12, 18, 24, and 48 hr, and MALAT-1 levels are determined by realtime-PCR. Exemplary primer sequences for detection are set forth as SEQID NOS:7 and 8. Using optimal concentrations and times, the effects ofthese compounds on selected HOTAIR-induced or -repressed genes are alsodetermined to further confirm that these compounds downregulate MALAT-1.

Role of ROS and ROS-dependent perturbation of the miR-27a:ZBTB10-Sp andmiR-20a/17-5p:ZBTB4-Sp pathways can also be further characterized. Usingthe same treatment protocol indicated above, the effects ofCDODA-Me/CF₃DODA-Me on ROS, miR-27a, ZBTB10, Sp1, Sp3, and Sp4 levelsare determined, and the effects of antioxidants (catalase, DTT, GSH,NAC) on this pathway and MALAT-1 expression are determined. Using anoptimal treatment time and concentration of CDODA-Me and CF₃DODA-Me,cells are transfected with miR-27a or miR-20a mimics or siZBTB10/siZBTB4(knockdown) to reverse the drug-induced downregulation of MALAT-1 and toconfirm the role of ROS-miR:ZBTB in mediating the repression of MALAT-1.In addition, the effects of the treatments on binding of Sp proteins andZBTB10 and ZBTB4 with the GC-rich MALAT-1 promoter are determined in aChIP assay according to established protocols.

The role of c-Myc in ROS-dependent repression of MALAT-1 can be furthercharacterized as described. The proposed studies coupled with theresults established by the inventors support the hypothesized linkagebetween drug-induced ROS and ROS-mediated effects on miR-ZBTB, whichresults in repression Sp-TFs and MALAT-1. A recent study (Lee, S. O., etal., “The Nuclear Receptor TR3Regulates mTORC1 Signaling in Lung CancerCells Expressing Wild-Type p53,” Oncogene 31:3265-3276, 2012)demonstrated that ROS(H₂O₂) induces chromatin shifts and relocalizesmethyl transferases/PRC2 from non-GC-rich to GC-rich areas and one ofthe repressed genes is Myc, which also regulates miR-27a andmiR-20a/17-5p (miR-17-92 cluster). It is demonstrated hereinabove thatCF3DODA-Me downregulates Myc protein expression in Panc1 cells. See FIG.10. Moreover, the inventors have observed comparable responses for otherROS inducers (not shown). Therefore, in an exemplary study, knockdown ofMyc by RNAi (siMyc) can determine the effects on miR:ZBTB-Sp axis andMALAT-1 expression, and a ChIP assay on the MALAT-1 promoter can be usedto confirm this pathway. ROS-induced relocalization of repressorcomplexes such as PRC2 will directly affect MALAT-1 transcription and aChIP assay can be used to determine changes in binding of Pol II,methylated histones (H3K27me3), and binding of PRC2 complex members onthe GC-rich MALAT-1 promote.

As described, the silencing of Sp1, or all three SpTFs, inhibited cellgrowth and induced apoptosis, which was comparable to effects observedafter knockdown of MALAT-1. Based on these studies, the prospectivestudies described herein are expected to demonstrate that MALAT-1 is acritical Sp-regulated lncRNA in pancreatic cancer. Further, thesestudies are predicted to show that drugs such as CDODA-Me and CF₃DODA-Metrigger a cascade which involves ROS-dependent disruption of miR-ZBTBcomplexes followed by ZBTB10/ZBTB4-mediated repression of MALAT-1.

In Vivo Investigations of MALAT-1 in Pancreatic Tumor Growth.

Mouse MALAT-1 (hcn) was identified as an overexpressed RNA incarcinogen-induced liver tumors, and the sequence homology between humanand mouse MALAT-1 was conserved. Accordingly, the role of MALAT-1 inpancreatic tumorigenesis can be investigated in both xenografts andtransgenic murine models. MALAT-1−/− mice have recently been generatedusing gene targeting and zinc finger nuclease approaches and, with theexception of some altered changes in gene expression in adult mice,these animals do not exhibit any histological or phenotypicabnormalities. In collaborative studies with the Texas Institute forGenomic Medicine (TIGM) at Texas A&M University, the inventors have usedgene-trapped ES cells to generate MALAT-1−/− mice. MALAT-1 expressionhas not been observed in tissues from these mice, and no phenotypic orhistological changes have been detected (data not shown). The MALAT-1−/−mice can be crossed with a transgenic mouse model expressing KRASG12Dwith a p53 mutation in the pancreas and 100% of these develop pancreatictumors. In addition, treatment of the transgenic mice with CF₃DODA-Mewill permit observation of the effects on expression of MALAT-1 andother genes/proteins involved in the miR-ZBTB-Sp-MALAT-1 pathway inpancreatic tumors.

The role MALAT-1 in regulating pancreatic tumor growth: exemplaryxenograft or orthotopic studies. Pancreatic cancer cells are transfectedwith a non-specific small hairpin oligonucleotide and an shMALAT-1 toobtain cells in which MALAT-1 is permanently silenced. These cells canbe used for xenograft experiments as described above. Control andMALAT-1-silenced cells are harvested by exposure to trypsin andresuspended in serum-free Hanks' balanced salt solution (HBSS).Viability is assessed by trypan blue exclusion, and only single-cellsuspensions exhibiting greater than 95% viability will be used. Forsubcutaneous tumors, tumor cells (1×10⁶ cells) suspended in a volume of200 μl are implanted subcutaneously in the flank of nude male animalsusing a 27-gauge needle. Tumors are allowed to grow unperturbed for10-14 d and when palpable tumors (200 mm3) first appear, mice arerandomly assigned to treatment or control groups. Mice are treated (10per treatment group) with placebo or CF3DODA-Me or CDODA-Me (2, 10, or20 mg/kg/d) (in corn oil) administered every second day for 4 to 6 weeks(depending on appearance and size of control tumors). L3.6pL cells areused for orthotopic pancreatic tumor studies. Cells are injecteddirectly into the pancreas and tumors are analyzed as previouslydescribed in this laboratory. Body, organ, and tumor weights aredetermined and tissues are used for histopathology.

Tumor sections from animals are prepared for in situ hybridization andimmunohistochemical analysis of proteins and in situ hybridization forSp and Sp-regulated gene products and mRNAs. Western blot analysis of Spand Sp-regulated genes are determined using tumor lysates. RNA extractedfrom control and treated tumors is used to confirm expression patternsfor identified MALAT-1, ZBTB10, miR-27a and MALAT-1-regulated genes.

Exemplary transgenic animal studies are described herein. Ras mutationsare common in human pancreatic ductal adenocarcinomas (PDACs) andoncogenic Ras^(G12D) mouse models coupled with other conditionaldeletions or mutations of p53, smad4 or TGFβRII. The inventors havedemonstrated that MALAT-1 silencing in Panc1 cells affected kinasesignaling (see FIGS. 13A and 13B). Accordingly, it is hypothesized thatloss of MALAT-1 in the Ras^(G12D) mice containing a p53 mutation willresult in a less severe or a delayed development of pancreatic cancer.

The role MALAT-1 in pancreatic tumor development can be addressed withthe following exemplary studies. MALAT-1-mice can be crossed with thePdxCre-expressing mice with both KrasG12D and Trp53R172H mutations. 100%of these animals develop pancreatic ductal adenocarcinomas. These doublemutant KrasG12D/p53mut mice in which MALAT-1 has been knocked out arepredicted to exhibit a delay in tumor occurrence and possibly somedecreased severity. After generation of the KrasG12D/p53mut-MALAT-1knockout mice, the development of pancreatic ductal adenocarcinoma(PDAC) is determined essentially as described (Hingorani, S. R., et al.,“Trp53R172H and KrasG12D Cooperate to Promote Chromosomal Instabilityand Widely Metastatic Pancreatic Ductal Adenocarcinoma in Mice,” CancerCell 7:469-483, 2005). Briefly, the development of cachexia, rapidweight loss or labored breathing and abdominal distension should becarefully monitored, and the animals are sacrificed when these symptomsare observed because these are end-points indicating advanced disease.Pancreatic tumor weights, volumes and histology are determined.Metastasis to the liver, lung, adrenals and peritoneum is determined byhistologic examination of each animal essentially accordingly toestablished procedures. Finally, expression of selected genes includingErbB1 (EGFR), ErbB2 and markers of epithelial to mesenchymal transition(e.g., β-catenin, N-cadherin, ZEB-1, ZEB-2, Snail, Slug, and relatedgenes) is determined.

Inhibition of tumor development/growth by CF3DODA-Me can be addressed.Previous studies have demonstrated that the triterpenoid anticanceragent Bartoxolone (CDDO-Me) increases the survival time ofKras^(G12)D/p53^(mut) mice from 20.5±0.9 wk (controls/unheated) to24.2±27 wk. In exemplary studies, randomized 4 week old transgenic mice(at least 10 per treatment group) are used. Corn oil control orCF3DODA-Me (10 or 25 mg/kg/d) in corn oil are administered by oralgavage every second day. The animals are monitored and analyzed asoutlined in above. Expression of MALAT-1, Sp1, Sp3, Sp4, ZBTB10, ZBTB4,and other selected Sp-regulated gene products are determined by realtime PCR and western blots.

Expected Results and Alternative Strategies. Results of xenograft andorthotopic models are expected to demonstrate that knockdown of MALAT-1in these cells result in a significant loss of their oncogenicpotential. A similar approach was used in the studies described abovewith regard to HOTAIR.

The following is a description of the regulation of HOTTIP (lncRNA) bySpTFs.

Introduction:

It is demonstrated hereinabove that HOTAIR, a lncRNA, is regulated bySpTFs, and that compositions that suppress SpTF expression also suppressHOTAIR expression. Therefore, the inventors investigated the regulationof other lncRNAs by SpTFs.

HOXA transcript at the distal tip (HOTTIP) is an lncRNA that influencesactivation of 5′ HOXA genes.

Results and Discussion:

The inventors investigated whether expression of HOTTIP is regulated bySpTFs, as is demonstrated above for HOTAIR. Using a combination ofsiRNAs for Sp1, Sp3, and Sp4 (iSp1/3/4), abundance of measurable HOTTIPmRNA was reduced in Panc1 cancer cells. See FIG. 16. Further, cellproliferation was assayed for Panc1 and L3.6pL pancreatic cancer cellsexposed to siRNA oligonucleotides for control siRNA (iCtrl), Sp1 (siSp1,SEQ ID NO:19), and HOTTIP (siHOTTIP, the sequence of which is set forthherein as SEQ ID NO:9). FIG. 17 demonstrates that both pancreatic cancercell lines exhibited similar reductions in cell growth with a knockdownof Sp1 and HOTTIP. Similarly, apoptosis assays were performed asdescribed above. Briefly, Panc1 and L3.6pL pancreatic cancer cells weretransfected with control siRNAs, siSp1 and siHOTTIP. After an incubationperiod, the cells were stained with Annexin V. Both Panc1 and L3.6pLcells exhibited increased staining, which is associated with inductionof apoptosis, upon knockdown of HOTTIP and Sp1, with staining especiallypronounced in Panc-1 cells (not shown). The effect of HOTTIP and Sp1knockdown on cell migration was assayed using a Boyden chamber assay asdescribed above, indicating that knockdown of each target significantlydecreased transwell migration (not shown). The effect of HOTTIP and Sp1knockdown on expression of various genes relevant to cancer progressionand control was also investigated. FIG. 18A demonstrates the effects ofRNAi knockdown of Sp1 and HOTTIP in Panc1 cells. As illustrated,knockdown of HOTTIP induced expression of cPARP, a marker for apoptosis,and decreased expression of Sp1-regulated genes EZH2 and Cyclin D1, aswell as survivin (a “cancer suppressor”) and VEGF (an angiogenic gene).Knockdown of HOTTIP in L3.6pL cells had similar effects, althoughknockdown of Cyclin D1 was less drastic. Knockdown of Sp1 followed asimilar pattern, except it did not induce an increase in cPARP in Panc1cells and consequence reduction in known Sp-regulated genes EZH2 andCyclin D1 was more complete in both cell lines. Finally, murinexenograft models were used as generally described in the abovedescriptions to assess the in vivo effects of HOTTIP on tumordevelopment. L3.6pL cells were transfected with siHOTTIP (or control)and later injected into nude mice. Tumor volumes were assessed over tendays. FIG. 19A demonstrates that knock down of HOTTIP resulted in asignificant reduction in tumor volume (i.e., prevented advancing tumorgrowth) compared to the control cells, with the major differencesapparent starting between 4 and 6 days. Mice were sacrificed and tumorweights were determined. FIG. 19B demonstrates that HOTTIP knockdownresulted in approximately a 75% reduction in tumor weight.

These results indicate that HOTTIP and Sp1 coordinately decrease severaltumor-suppressor qualities in cells, and their knockdown results insimilar responses, such as inhibition of cancer cell growth, inductionof apoptosis, and inhibition of migration. HOTTIP, via its interactionwith Sp1, is thus a promising target for control or treatment of cancerssuch as pancreatic cancer.

The following is a description of the regulation of HULC and otherlncRNAs by SpTFs in liver and cervical cancer cells.

Introduction:

It is demonstrated hereinabove that HOTAIR, a lncRNA, is regulated bySpTFs, and that compositions that suppress SpTF expression also suppressHOTAIR expression. Therefore, the inventors investigated the regulationof other lncRNAs by SpTFs in cancer cell types other than pancreaticcancer.

Results and Discussion:

The inventors transfected two lines of liver cancer cells (HEPG2 andSK-Hep1) and cervical cancer cells (HeLa) with siRNAs for the Sp1transcription factor (siSp1) according to standard protocols describedherein, to determine whether this TF plays a pro-oncogenic role asobserved in other cancer cell types, including pancreatic cancer cells.RNAi knockdown using siSp1 significantly decreased cell proliferation,as expressed as relative percent cell viability, in both liver cancercell types as well as HeLa cells at both 3 and 5 days post transfection(not shown). Apoptosis assays were performed on SK-Hep1 cells using theAnnexin V staining (described above). Briefly, cells were transfectedwith siSp1, and were subsequently stained with Annexin V, whose uptake(thus staining) is correlated with apoptosis. Knockdown of Sp1 inSK-Hep1 cells resulted in a marked increase in apoptosis (not shown).Furthermore, transwell migration of Hela and SK-Hep1 cells was assayedin a Boyden chamber assay (not shown), indicating a significant decreasein both cell types upon knockdown of Sp1 (not shown). Similarly, Sp1knockdown was demonstrated to significantly decrease invasion capacityof SK-Hep1 cells (not shown). Using a scratch wound-healing assay, whereadherent cells are scraped away from the culture surface and allowed toreestablish adherence over time, it was demonstrated that SK-Hep1 cellsare slower to reestablish, i.e., have lower motility, after knockdown ofSp1. These data derived from liver and/or cervical cancer cells areconsistent with prior observations regarding the pro-oncogenic role ofSp1 in other cancer cells, indicating the transcription factor's role inregulating cell growth, resisting apoptosis, and promoting migration,invasion and motility.

After establishing the pro-oncogenic role of Sp1 in liver and cervicalcancer cells, the inventors examined the role of Sp in the expression ofvarious lncRNAs in these cancer cells and the roles of certain lncRNAsin these cancer cells. FIGS. 20A-20C demonstrate that lncRNAs, such asAY129027 (Yang et al., Hepatology 54:1679-1689 (2011)), HULC (Panzitt etal., Gastroenterology 132: 330-342 (2007)), and linc-HEIH (Yang et al.,Hepatology 54:1679-1689 (2011)) are decreased when Sp1 is knocked downby RNAi (using siSp1). HULC lncRNA was detected and quantified usingforward and reverse oligonucleotide primers whose sequences are setforth herein as SEQ ID NOS:13 and 14, respectively. AY129027 lncRNA wasdetected and quantified using forward and reverse oligonucleotideprimers whose sequences are set forth herein as SEQ ID NOS: 15 and 16,respectively. linc-HEIH lncRNA was detected and quantified using forwardand reverse oligonucleotide primers whose sequences are set forth hereinas SEQ ID NOS: 17 and 18, respectively. Specifically, FIG. 20A showsthat AY129027 and HULC RNA levels significantly declined in HepG2 cells72 hours after transfection with siSp1. Similarly, FIG. 20B shows thatAY129027 and HULC RNA levels significantly declined in HeLa cells 72hours after transfection with siSp1. Finally, FIG. 20C shows thatAY129027, linc-HEIH and HULC RNA levels significantly declined inSK-Hep1 cells 72 hours after transfection with siSp1, in addition toHOTAIR RNA levels.

After establishing that Sp1 regulates several lncRNAs in these cancercells, specific focus was applied on HULC as a model lcnRNA forcharacterization in liver and cervical cancer cells. As a preliminarymatter, FIGS. 21A-21C respectively demonstrate that RNAi using siHULCeffectively and significantly decreases HULC lcnRNA levels in HepG2,HeLa, and SK-Hep1 cells by 72 hours post transfection. The sequence ofsi HULC is set forth herein as SEQ ID NO:12. While HULC knockdown usingsiHULC did not affect the relative cell viability of HepG2 cells (FIG.22A), both HeLa and SK-Hep1 cells exhibited significant reductions incell proliferation at 5 and 7 days post transfection (FIGS. 22A and 22B,respectively). Using the Annexin V staining approach described above,RNAi knockdown of HULC in SK-Hep1 cells using siHULC resulted in ademonstrable increase in apoptosis (not shown). Finally, FIGS. 23A and23B demonstrate that RNAi knockdown of HULC using siHULC in SK-Hep1cells, a highly invasive cell line, resulted in a significant decreasein invasion/migration as determined in a Boyden chamber assay.

Drugs known to suppress SpTFs were also tested for their ability tocause suppression of HULC lncRNA in liver and cervical cancer cells.FIG. 24A demonstrates that application of CF₃DODA, an establishedSp-targeting compound (see discussion above), significantly decreasedSp1, AY129027 and HULC expression levels in HepG2 and HeLa cells asdetermined 48 hrs after exposure.

In conclusion, it is demonstrated that the SpTF Sp1 maintains itspro-oncogenic characteristics in liver and cervical cancer cells.Moreover Sp1 regulates the expression of multiple lncRNAs in thesecells, as determined by RNAi knockdown assays. One representativelncRNA, HULC, is demonstrated to promoter tumorigenic characteristics,such as invasion, apoptosis suppression, and invasion, as determined byRNAi knockout. Finally, it is established that drugs suppressing Sp1activity expression also suppress HULC expression. This demonstratesthat Sp1 and additional pro-oncogenic lncRNAs coordinately decreaseseveral tumor-suppressor qualities in cells, and their knockdown resultsin similar responses, such as inhibition of cancer cell growth,induction of apoptosis, and inhibition of migration, in a variety ofcancer cells. Thus, via their interactions with Sp1, pro-oncogeniclncRNAs are promising targets for control or treatment a variety ofcancers.

The following is a description of the regulation of oncogenic MiRexpression by SpTFs.

Introduction:

It is demonstrated above that numerous oncogenic lncRNAs are regulatedby SpTFs, and thus, serve as targets for drugs effective to downregulateSpTFs. MicroRNAs (miR) are another class of ncRNAs. Many miRs areoncogenic and are overexpressed in different tumor types. OncogenicmiR-17, miR-20a, miR-106b, miR-93, and miR-106a are members of theoncogenic miR-17-92, miR-106a-363, and miR-106b-25 clusters. The role ofSpTFs in regulating the expression of miR was investigated.

Methods:

siRNAs for Sp1, Sp3, Sp4 and LMN (Lamin) were purchased fromSigma-Aldrich.

Panc28 and L3.6pL pancreatic cancer cell lines were seeded (6×10⁴ perwell) in 6-well plates in DMEM:Ham's F-12 medium supplemented with 2.5%charcoal-stripped FBS without antibiotic and left to attach for 1 d.Single and triple Sp siRNA knockdown (iSp1, iSp3, iSp4) along withiLamin as control was performed using LipofectAMINE™2000 transfectionreagent as per the manufacturer's protocol to attain 100 nMconcentration of siRNA in the media. Cells were left with thetransfection mixture for 24-48 hrs before changing to fresh 2.5%DMEM-F12 media. Cells were harvested after 60-72 hrs for protein, mRNAor miRNA. miRNA was extracted using mirVana™ miRNA extraction kit(Applied Biosystems) according to manufacturer's protocol and was usedto assess expression of various non-coding RNAs using different primersand determined by real-time PCR.

Results and Discussion:

Knockdown of Sp1 also significantly decreased their expression in breastcancer cells (FIG. 9D). MiR-21 and miR-181b have previously beencharacterized as oncogenic miRs that play an important role in tumordevelopment and growth (Cancer Res 70:4528 (2010); Clin Exp Metastasis28:27 (2011); Oncogene 29:1787 (2010)). Results illustrated in FIGS.25A-25D demonstrate that knockdown of Sp1 (iSp1), Sp3 (iSp3), Sp4(iSp4), and their combination (iSp1/3/4) decrease expression ofoncogenic miR-21 and miR-181b in Panc28 and L3.6pL pancreatic cancercells.

These results demonstrate that the oncogenic small ncRNAs (i.e., miRs)are also regulated by SpTFs, and therefore, are also targets for drugsthat downregulate SpTFs.

While the preferred embodiment of the invention has been illustrated anddescribed, it will be appreciated that various changes can be madetherein without departing from the spirit and scope of the invention.

1. A method of inhibiting growth of a cell characterized byoverexpression of at least one specificity protein (Sp)-regulatednon-coding RNA (ncRNA) and expression of at least one Sp transcriptionfactor (SpTF), the method comprising contacting the cell with aneffective amount of an SpTF agent.
 2. The method of claim 1, wherein thecell is a cancer cell.
 3. The method of claim 2, wherein the cancer cellis selected from the group consisting of a breast cancer cell, apancreatic cancer cell, a liver cancer cell, a lung cancer cell, aprostate cancer cell, and a follicular lymphoma cancer cell.
 4. Themethod of claim 2, wherein the overexpression of the at least oneSp-regulated ncRNA in the cancer cell can be determined by comparing theexpression level in the cancer cell to a reference standard.
 5. Themethod of claim 2, wherein comparing the expression level in the cancercell to a reference standard comprises comparing the expression level ofthe at least one Sp-regulated ncRNA to the expression level of the atleast one Sp-regulated ncRNA in a noncancerous cell derived from thesame tissue.
 6. The method of claim 1, wherein the at least oneSp-regulated ncRNA is a long non-coding RNA (lncRNA).
 7. The method ofclaim 6, wherein the lncRNA is selected from the group consisting ofHOTAIR, HOTAIRM, HOTTIP, MALAT-1, linc-HEIH, HULC, and AY12907.
 8. Themethod of claim 1, wherein the at least one SpTF is Sp1, Sp3, Sp4 orother Sp/KLF transcription factor.
 9. The method of claim 1, wherein theSpTF agent comprises: (i) a phytochemical or derivative that inducesreactive oxygen species (ROS) or phosphatase activity; (ii) anaturally-occurring or synthetic triterpenoid; (iii) a non-steroidalanti-inflammatory drug (NSAID); (iv) an antisense microRNAoligonucleotide; (v) an agent that causes overexpression of ZBTB10,ZBTB4, or related transcriptional repressor, or that inducesproteasome/caspase-dependent degradation of Sp transcription factors;(vi) a thiazolidinedione; (vii) a nitro-aspirin; (viii) anisothiocyanate; (ix) aspirin; (x) arsenic trioxide; (xi) metformin;(xii) silibinin; or (xiii) a cannabinoid.
 10. The method of claim 9,wherein the triterpenoid is methyl2-cyano-3,12-dioxooleana-1,9-dien-28-oate or methyl2-cyano-3,11-dioxo-18β-olean-1,12-dien-30-oate.
 11. The method of claim1, further comprising contacting the cell with one or more smallinterfering RNA (siRNA) molecules that hybridize with the mRNA encodingan SpTF under physiological or cell-culture conditions.
 12. The methodof claim 1, wherein the cell is in vivo in a subject.
 13. The method ofclaim 1, wherein the cell is in vitro.
 14. The method of claim 13,wherein the cell is derived from or comprised in a sample obtained froma subject having a cell proliferative disease or suspected of having acell proliferative disease.
 15. A method of treating a cellproliferative disease, the method comprising administering to a subjectin need a therapeutically effective amount of an specificity proteintranscription factor (SpTF) agent, wherein the subject has at least onetransformed cell characterized by the overexpression of at least onespecificity protein (Sp)-regulated non-coding RNA (ncRNA) and theexpression of at least one SpTF.
 16. The method of claim 15, wherein theSpTF agent is comprised in a pharmaceutically acceptable composition.17. The method of claim 15, wherein the subject is selected from thegroup consisting of: human, monkey, horse, cow, sheep, goat, dog cat,mouse, rat, and guinea pig.
 18. A method of predicting the response of asubject with a cell proliferative disease to a specificity proteintranscription factor (SpTF) agent-based treatment, the methodcomprising: (i) determining the expression level of at least onespecificity protein (Sp)-regulated non-coding RNA (ncRNA) in a cellsample obtained from the subject, (ii) determining the expression statusof at least one specificity protein transcription factor (SpTF) in thesame or similar cell sample obtained from the subject, and (iii)predicting a positive response of the subject with a cell proliferativedisease to treatment with an SpTF agent when the at least oneSp-regulated ncRNA is overexpressed in the cell sample and the SpTF isexpressed in the same or similar cell sample.
 19. The method of claim18, wherein overexpression of the at least one Sp-regulated ncRNA isdetermined by comparing the expression of the Sp-regulated ncRNA in thecell sample to a reference standard.
 20. The method of claim 19, whereincomparing the expression of the Sp-regulated ncRNA in the cell sample toa reference standard comprises comparing the expression level of the atleast one Sp-regulated ncRNA to the expression level of the at least oneSp-regulated ncRNA in a noncancerous cell derived from the same tissue.21. The method of claim 18, further comprising administering an SpTFagent to the subject that is predicted to have a positive response totreatment.
 22. A method of monitoring the efficacy of a specificityprotein transcription factor (SpTF) agent-based treatment in a subjectwith a cell proliferative disease, the method comprising: (i)determining the expression level of at least one specificity protein(Sp)-regulated non-coding RNA (ncRNA) in a first cell sample obtainedfrom the subject, (ii) administering at least one SpTF agent to thesubject, and (iii) determining the expression level of the at least oneSp-regulated ncRNA in a second cell sample obtained from the subject,wherein the second cell sample is obtained from the same or similar cellsource within the subject, wherein the second cell sample is obtainedfrom the subject at a time after the first cell sample is obtained andafter the at least one SpTF agent is administered to the subject,wherein the treatment is determined to be effective when the expressionlevel of the Sp-regulated ncRNA is lower in the second sample than inthe first sample.
 23. A method of evaluating a candidate specificityprotein transcription factor (SpTF) agent for use in treatment of a cellproliferative disease, the method comprising: (i) contacting a candidateSpTF agent to a cell characterized by: (a) overexpression of at leastone specificity protein (Sp)-regulated non-coding RNA (ncRNA), and (b)expression of an SpTF, (ii) determining the expression level of the atleast one Sp-regulated ncRNA subsequent to the contacting step (i), and(iii) comparing the expression level of the at least Sp-regulated ncRNAin step (ii) to a reference standard, wherein a reduced expression levelof the at least Sp-regulated ncRNA in step (ii) in comparison to thereference standard is indicative of the efficacy of the candidate SpTFagent for treatment of a cell proliferative disease.
 24. The method ofclaim 23, wherein the cell is a cancer cell and the reference standardcomprises a noncancerous cell derived from the same tissue as the cancercell.